Bordetella bronchiseptica attenuated strain HN60 and application thereof

Abstract

The invention belongs to the technical field of biological pharmacy, and particularly provides a bordetella bronchiseptica attenuated strain HN60 and application thereof. The preservation number of the bordetella bronchiseptica attenuated strain HN60 is CCTCC NO: M20221719, and the bordetella bronchiseptica attenuated strain HN60 is obtained by culturing a bordetella bronchiseptica wild strain HN03, has stable biological characteristics, has good immunogenicity, is safe and reliable, and has positive prevention and efficient control effects on infectious tracheobronchitis of dogs.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals, and specifically provides a Bordetella bronchiseptica attenuated strain HN60 and an application thereof. Background Art

[0002] Canine infectious tracheobronchitis (kennel cough) is an important disease in dogs. It often occurs and spreads in groups of dogs raised in groups, such as kennels, stray dog ​​shelters, pet shops and pet hospitals. It is caused by infection with Bordetella bronchiseptica (Bb). Dogs of different breeds, genders and ages can be infected with this disease, but young dogs are the most sensitive and the harm is more serious. Clinically, paroxysmal cough, tracheobronchitis and rhinitis are the main symptoms.

[0003] Bordetella bronchiseptica was first isolated from the respiratory tract of dogs with canine distemper. It is a Gram-negative coccobacillus belonging to the genus Bordetella. It can infect a variety of mammals, causing atrophic rhinitis and pneumonia in pigs, bronchial pneumonia in rabbits and guinea pigs, and respiratory infections in cats, mice and horses. It is highly contagious to dogs and can be easily transmitted through direct contact between dogs.

[0004] There are four phenotypes of Bordetella bronchiseptica growing on the Bauer blood plate, namely, phase I, phase II, phase III and rough type. The colony morphology of phase I bacteria on the Bauer blood plate is small pearl-like colonies, some strains are hemolytic, and express a series of virulence factors. The colony morphology of phase III bacteria on the Bauer blood plate is large gray-white flat colonies, which are not hemolytic and do not express corresponding virulence factors. Studies have shown that phase I strains are highly virulent strains, and other variant strains are weak strains.

[0005] Vaccination plays an important role in preventing infectious canine tracheobronchitis. The present invention selects a wild strain of Bordetella bronchiseptica isolated in China for cultivation to obtain a weakened strain, which has an active prevention and efficient control effect on canine infectious tracheobronchitis.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The first object of the present invention is to provide a Bordetella bronchiseptica attenuated strain HN60.

[0008] The second object of the present invention is to provide the application of the Bordetella bronchiseptica attenuated strain HN60.

[0009] The third object of the present invention is to provide a vaccine.

[0010] The fourth object of the present invention is to provide a method for preparing the above-mentioned vaccine.

[0011] The fifth object of the present invention is to provide a highly virulent strain of Bordetella bronchiseptica HN03.

[0012] The sixth object of the present invention is to provide an application of the above-mentioned Bordetella bronchiseptica virulent strain HN03.

[0013] In order to achieve the above object, the present invention particularly adopts the following technical solutions:

[0014] A weak strain of Bordetella bronchiseptica HN60, with a preservation number of CCTCCNO:M20221719, preserved in the China Center for Type Culture Collection, with a preservation address of Wuhan University, Wuhan, China, and a preservation date of November 3, 2022.

[0015] Application of the Bordetella bronchiseptica attenuated strain HN60 or its culture in the preparation of a drug for preventing Bordetella bronchiseptica infection.

[0016] Furthermore, the drug is a vaccine.

[0017] Preferably, the subject is a dog or a cat.

[0018] A vaccine comprises an immunologically effective amount of Bordetella bronchiseptica attenuated strain HN60 or its culture, and pharmaceutically acceptable adjuvants.

[0019] Furthermore, the Bordetella bronchiseptica attenuated strain HN60 content is ≥1×10 6 CFU / head portion; preferably, the Bordetella bronchiseptica attenuated strain HN60 content is 1×10 6 ~1×10 10 CFU / head portion; more preferably, the Bordetella bronchiseptica attenuated strain HN60 content is 1×10 9 CFU / head.

[0020] Furthermore, the Bordetella bronchiseptica HN60 culture is a culture of 1 to 60 generations.

[0021] Furthermore, the pharmaceutically acceptable excipient is a lyoprotectant, which includes SPGA, sugars, gelatin, protein, a substance containing protein or / and a buffer.

[0022] Preferably, the sugar is selected from sorbitol, mannitol, starch, sucrose, glucose or dextran; the protein is selected from albumin or casein; the substance containing protein is bovine serum or skim milk; and the buffer is phosphate buffer.

[0023] Preferably, the lyoprotectant is 20 w / v% sucrose and 2 w / v% gelatin.

[0024] Preferably, the volume ratio of the Bordetella bronchiseptica attenuated strain HN60 or its culture to the pharmaceutically acceptable excipient is 1:1.

[0025] The preparation method of the vaccine comprises the following steps: uniformly mixing the Bordetella bronchiseptica attenuated strain HN60 or its culture with pharmaceutically acceptable auxiliary materials to obtain the vaccine.

[0026] A strong strain HN03 of Bordetella bronchiseptica, which is homologous to the weak strain HN60 of Bordetella bronchiseptica, with a preservation number of CCTCCNO:M20221718. It is preserved in the China Center for Type Culture Collection, with a preservation address of Wuhan University, Wuhan, China, and a preservation date of November 3, 2022.

[0027] The application of the Bordetella bronchiseptica virulent strain HN03 in testing the efficacy of Bordetella bronchiseptica vaccines.

[0028] Compared with the prior art, the technical effects of the present invention are:

[0029] The invention selects a wild strain HN03 of Bordetella bronchiseptica isolated in China for cultivation, and obtains a weakened strain HN60, which has stable biological characteristics, good immunogenicity, is safe and reliable, and has an active prevention and efficient control effect on canine infectious tracheobronchitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 It is the comparison of the nucleotide sequences of the original strain and the attenuated strain BvgS in Example 2 (cdh: attenuated strain, cdq: original strain);

[0032] Figure 2 It is the amino acid sequence alignment of the original strain and the attenuated strain BvgS in Example 2 (cdh: attenuated strain, cdq: original strain). DETAILED DESCRIPTION

[0033] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] The term "immunologically effective amount" refers to the amount necessary to exert their immunological effect in the subject to which the vaccine is administered without causing undue side effects. The exact amount of the ingredients used and the vaccine to be administered will vary depending on factors such as the type of disease being treated, the type and age of the animal being treated, the mode of administration, and the other ingredients in the vaccine.

[0035] The term "pharmaceutically acceptable excipient" refers to an excipient that does not irritate the body and does not interfere with the biological activity and properties of the compound being used.

[0036] The term "prevention" as used herein refers to all actions to inhibit Bordetella bronchiseptica infection or delay the onset of the disease and reduce bacterial excretion after infection by administering the vaccine of the present invention.

[0037] The present invention provides a Bordetella bronchiseptica attenuated strain HN60 (Bordetellabronchiseptica HN60), with a preservation number of CCTCCNO: M20221719, which is preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, and the preservation date is November 3, 2022.

[0038] The attenuated strain HN60 of Bordetella bronchiseptica has stable biological characteristics, good immunogenicity, safety and reliability. The prepared attenuated live vaccine can produce a high level of antibodies and has a good protective effect against the strong toxin attack of Bordetella bronchiseptica.

[0039] The pathogenicity test showed that the pathogenicity of Bordetella bronchiseptica HN60 to dogs decreased significantly between the 1st and 60th generations. The dogs did not show clinical symptoms after 14 days of observation after the 60th generation of inoculation. Therefore, compared with its parent, the highly virulent Bordetella bronchiseptica HN03, the pathogenicity of this strain is significantly reduced, and it is a weak strain.

[0040] Immunogenicity tests showed that Bordetella bronchiseptica HN60 cultured to the 60th generation still had good immunogenicity. Dogs could resist the attack of the highly virulent Bordetella bronchiseptica HN03 28 days after vaccination. At the same time, dogs that were not vaccinated with Bordetella bronchiseptica HN60 culture could not resist the attack of Bordetella bronchiseptica HN03 and all became ill.

[0041] The virulence reversion test showed that Bordetella bronchiseptica HN60 cultured between the 1st and 60th generations did not revert to virulence after continuous contact and passage in the dog group after inoculation. Therefore, after Bordetella bronchiseptica HN60 was inoculated in the dog group, it would not re-evolve into a virulent virus and cause disease, and its safety was guaranteed.

[0042] Compared with its parent, the highly virulent Bordetella bronchiseptica HN03, the cultures of Bordetella bronchiseptica HN60 from the 1st to the 60th generations commonly showed a nucleotide base G inserted into the BvgS gene at the 2004 position, resulting in a frameshift mutation of the BvgS gene after the insertion site. The toxicity is lower than that of the parental highly virulent strain, and it has genetic stability.

[0043] The common characteristic changes in viral genes of different generations of Bordetella bronchiseptica HN60 cultures are most likely the reason for the reduction in virulence of its parental virulent strain, and these changes did not lead to changes in its immunogenicity.

[0044] The Bordetella bronchiseptica attenuated strain HN60 or its culture is used for preparing medicine for preventing Bordetella bronchiseptica infection.

[0045] In some embodiments, the functional component in the drug is a live whole bacterial antigen of the attenuated strain HN60 of Bordetella bronchiseptica or its culture; and the amount of the functional component in the drug is an immune effective amount.

[0046] In some embodiments, the drug may be a vaccine, and the drug subject may be a dog or a cat.

[0047] The vaccine of the present invention comprises an immune effective amount of Bordetella bronchiseptica attenuated strain HN60 or its culture, and pharmaceutically acceptable adjuvants.

[0048] In one embodiment, the immune effective amount refers to a content of attenuated strain HN60 of Bordetella bronchiseptica of ≥1×10 6 CFU / head, preferably 1×10 6 ~1×10 10 CFU / head, more preferably 1×10 9 CFU / head.

[0049] In one embodiment, the Bordetella bronchiseptica HN60 culture is a culture of passages 1 to 60. For example, it can be selected from a culture of passages 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, or 60.

[0050] In some embodiments, the pharmaceutically acceptable excipient is a lyoprotectant, which includes SPGA (a lyoprotectant commonly used in the art, composed of sucrose, phosphate, glutamic acid and albumin), sugars, gelatin, proteins, protein-containing substances or / and buffers; sugars are preferably selected from sorbitol, mannitol, starch, sucrose, glucose or dextran; proteins are preferably selected from albumin or casein; protein-containing substances are preferably bovine serum or skim milk; buffers are preferably phosphate buffers; lyoprotectants are preferably 20w / v% sucrose and 2w / v% gelatin. In particular, when such lyoprotectants are added to vaccines, the vaccines are very suitable for freeze-drying. Therefore, in a more preferred form of this embodiment, the live attenuated vaccine is in a freeze-dried form.

[0051] In some embodiments, the volume ratio of the Bordetella bronchiseptica attenuated strain HN60 or a culture thereof to the pharmaceutically acceptable excipient is 1:1.

[0052] The present invention also provides a Bordetella bronchiseptica virulent strain HN03 (Bordetellabronchiseptica HN), which is homologous to the Bordetella bronchiseptica weak strain HN60, with a deposit number of CCTCCNO: M20221718, and is deposited in the China Center for Type Culture Collection, with a deposit address of Wuhan University, Wuhan, China, and a deposit date of November 3, 2022. The Bordetella bronchiseptica virulent strain HN03 can be used to test the efficacy of Bordetella bronchiseptica vaccines.

[0053] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0054] Example 1 Isolation and Identification of Bordetella bronchiseptica in Dogs

[0055] 1.1 Isolation and identification of Bordetella bronchiseptica in dogs

[0056] Wild strains of Bordetella bronchiseptica were isolated from dogs clinically infected with Bordetella bronchiseptica. The clinical symptoms of infected animals included coughing, sneezing, runny nose, etc., which were used as the criteria for selecting animals.

[0057] Nasal or throat swabs were collected aseptically from dogs with clinical symptoms, and the collected swabs were inoculated on Bjornsberg blood agar plates and MacConkey agar plates, respectively, and cultured at 37°C for 40 to 48 hours. The colony morphology was observed, and typical colonies were picked for subculture, purification, and Gram staining microscopy. The results showed that 13 suspected Bordetella bronchiseptica colonies were isolated from 123 canine nasopharyngeal swabs collected, that is, small pearly, milky white colonies grown on Bjornsberg blood agar plates for 40 to 48 hours, and some strains had β-hemolysis; when cultured on MacConkey plates at 37°C for 40 to 48 hours, the colony morphology was colorless or tea-yellow with neat edges, smooth and moist, and smaller colonies. Gram staining microscopy of the 13 suspected isolated bacteria were all negative for cocci.

[0058] The above-mentioned isolated strains were identified by the Bordetella bronchiseptica PCR identification method reported by Hozbor, D. (Hozbor, D., F. Fouque, and N. Guiso, Detection of Bordetella bronchiseptica by the polymerase chain reaction. Research in Microbiology, 1999. 150 (5): 333-341.), and the results showed that all 13 suspected strains amplified a specific band of 237 bp in size, and PCR identification showed that they were all Bordetella bronchiseptica. According to the report of Bai Xue et al. (Bai Xue, et al., Isolation and Identification of Bordetella bronchiseptica in Dogs. Animal Husbandry and Veterinary Medicine, 2010. 42 (2): 55-58.), the isolated strains were biochemically identified, and the results showed that the biochemical test results of the 13 isolated strains were consistent with the biochemical characteristics of Bordetella bronchiseptica.

[0059] The above results confirmed that the isolated pathogenic strain was a wild strain of Bordetella bronchiseptica canine.

[0060] 1.2 Screening of highly virulent strains of Bordetella bronchiseptica in dogs

[0061] 1.2.1 Virulence to mice The isolates were cultured on TSA containing 10% newborn calf serum at 37°C for 24 h, and then the culture was suspended in PBS (0.01 mol / L, pH 7.2) to prepare a plate with a viable bacterial content of 5 × 10 8 CFU / mL of bacterial suspension. The bacterial suspension was injected intraperitoneally into BALB / c mice, with the injection volume of 0.2 mL (1×10 8CFU / each) and observed for 10 days after the challenge. The results showed that the challenge dose of BJ01, HN01, HN03 and HN04 was 1×10 8 CFU / mouse, it can cause the death of all mice, while the other strains cannot cause all the death.

[0062] 1.2.2 LD for mice 50 The LD of four strains of Bordetella bronchiseptica, BJ01, HN01, HN03 and HN04, against mice were determined. 50 Specifically, when the bacterial count is 10 9 CFU / mL, and make 10-fold serial dilutions to 10 7 CFU / mL, the bacterial solution of different dilutions was intraperitoneally injected into 8 16-18g BALB / c mice, each group was kept in isolation, and the mice were observed for 10 days after inoculation. The mortality of mice in each group was recorded, and the LD to mice was calculated by Karber method. 50 The mice were challenged with BJ01, HN01, HN03 and HN04 strains respectively, and the LD 50 The results showed that the LD of BJ01, HN01, HN03 and HN04 strains in mice 50 They are 1.51×10 7 CFU / unit, 3.87×10 7 CFU / unit, 1.43×10 7 CFU / cell and 2.37×10 7 CFU / mouse, the results showed that HN03 strain was the most virulent to mice.

[0063] 1.3 Toxicity test on dogs

[0064] The HN03 strain, which was the most virulent to mice, was tested for virulence to dogs. The HN03 strain was cultured on TSA containing 10% newborn calf serum at 37°C for 24 h, and then the culture was suspended in PBS (0.01 mol / L, pH 7.2) to prepare a viable bacterial cell culture solution with a viable bacterial content of 1×10 9 CFU / mL of bacterial suspension. Healthy susceptible dogs with negative Bb antigen and antibody were inoculated with bacterial suspension by nasal drops, with an inoculation volume of 2 mL for each dog. The dogs were observed for 14 consecutive days after the challenge, and the clinical symptoms of the test dogs after inoculation were observed. The results showed that 3 / 3 of the dogs inoculated with HN03 strain had sneezing, 2 / 3 had coughing, and 3 / 3 had eye secretions and runny nose, which could cause typical clinical symptoms in the inoculated dogs. The canine bronchiseptica Bordetella HN03 strain was submitted for preservation as a weakened original strain.

[0065] Example 2 Cultivation of a canine bronchiseptica Bordetella attenuated strain

[0066] 2.1 Cultivation of a low-virulence strain of Bordetella bronchiseptica in dogs

[0067] 2.1.1 Cultivate canine bronchiseptica Bordetella HN03 strain by streaking on Baojiang's blood agar plates and culturing at 42°C for 5 to 7 days per generation. Pick out large gray, flat colonies and continue to subculture according to the above method. Continue to subculture according to the above method until the 60th generation. The colony morphology is large gray, flat colonies, which agglutinate with O factor serum but not with K factor serum, and are phase III bacteria.

[0068] 2.1.2 Safety of strains The bacterial solution prepared by high temperature culture for 60 generations was inoculated into 5 healthy susceptible dogs aged 6 to 8 weeks, with 2 ml / dog (containing 2×10 10 CFU live bacteria), and another 5 dogs were taken as the control group, each of which was inoculated with 2 ml of BHI medium (Brain-Heart Infusion Broth) and observed for 14 days. The inoculated dogs' spirit, feeding, and clinical symptoms such as coughing, sneezing, vomiting, eye secretions, and nasal secretions were observed. The body temperature of the experimental dogs was measured before and after inoculation. The results showed that after the 60th generation of bacteria were inoculated in dogs, 5 / 5 of them showed no abnormality. The results showed that the F60 generation of Bordetella bronchiseptica HN03 strain was safe. From the 60th generation onwards, the Bordetella bronchiseptica HN03 strain was renamed as HN60 strain and submitted for deposit.

[0069] 2.2 Genetic characteristics of bacterial strains

[0070] The original strain HN03 before attenuation and the attenuated strain HN60 were sequenced for their whole genomes, and the sequencing results showed that the BvgS gene of the attenuated strain HN60 had a nucleotide base G inserted at the 2004 position, resulting in a frameshift mutation in the BvgS gene after the insertion site. Figure 1 and attached Figure 2 .

[0071] 2.3 Genetic stability

[0072] The prepared Bordetella bronchiseptica HN60 strain of the present invention was streaked and cultured on a Baojiang blood plate, cultured in a 37°C incubator for 40 to 48 hours, and then a single colony was picked from the cultured plate and placed on a Baojiang blood plate, and the colony morphology was observed for 60 consecutive passages. The results showed that the colonies of each passage for 60 generations were large gray, flat colonies without variation. The O factor and K factor serum agglutination tests were performed on the 1st, 10th, 20th, 30th, 40th, 50th and 60th generation cultures, respectively. The colonies of different generations all agglutinated with the O factor serum, and did not agglutinate with the K factor serum, and were all phase III bacteria. At the same time, the primers BvgS-F / BvgS-R (see Table 1) were used to extend and sequence the 1st, 10th, 20th, 30th, 40th, 50th and 60th generation templates of the HN60 strain, and the sequencing results showed that the site of the strains of different generations did not change, and could be stably inherited.

[0073] Table 1 Sequencing primer sequences Primer name Primer sequences BvgS-F 5'-CATTTCGAACGACGAACTCGCCT-3' BvgS-R 5'-CAATGTCCAGGATGTCGCCTATC-3'

[0074] 2.4 Virulence reversion test

[0075] 2.4.1 Preliminary test of toxicity reversion

[0076] The viable bacterial content of Bordetella bronchiseptica HN60 strain culture was adjusted to 1×10 10 CFU / ml, 12 healthy susceptible dogs were inoculated with a dose of 2ml / dog, containing 2×10 10 CFU / ml. The 12 vaccinated dogs were randomly divided into 6 groups, with 2 dogs in each group. Two dogs were dissected on the 3rd, 5th, 7th, 9th, 11th and 13th days after vaccination, and tonsils, submandibular lymph nodes, tracheal lymph nodes, lungs, liver, spleen, heart, kidneys, larynx, trachea and daily nasal swabs were taken. The content of Bordetella bronchiseptica in nasal swabs and organ tissues was detected by live bacteria counting method. At the same time, 2 blank dogs were set up without vaccination. Each tissue was dissected and the content of Bordetella bronchiseptica in each tissue was detected. The temperature was measured 3 days before vaccination and every day after vaccination. The results showed that there was no abnormality in body temperature after vaccination. See Table 2 for details. The clinical symptoms of the vaccinated dogs were observed every day after vaccination, and no abnormalities were found.

[0077] Table 2 Body temperature of test dogs before and after vaccination (℃)

[0078] Note: “ / ” means the experimental dogs were autopsied but body temperature was not measured.

[0079] Nasal swabs were collected daily before and after vaccination for live bacteria counts. The results are shown in Table 3. The results showed that the nasal swab content was the highest 2 days after vaccination, and then it showed a downward trend. By 10 days after vaccination, no Bordetella bronchiseptica could be detected in the nasal swabs. The vaccinated dogs were autopsied at different times after vaccination, and no abnormalities were found in any organs. The live bacteria counts of Bordetella bronchiseptica in each organ showed that the Bb content in the submandibular lymph nodes and organ lymph nodes was the highest among the organs, and its content was much higher than the Bordetella bronchiseptica content in the nasal swabs. Among them, the Bordetella bronchiseptica content in the submandibular lymph nodes and tracheal lymph nodes was the highest 7 days after vaccination, as shown in Table 4. Based on the above results, it was determined that the target tissues for virulence reversion were the submandibular lymph nodes and tracheal lymph nodes, and the collection time was 7 days after vaccination.

[0080] Table 3 Results of Bordetella bronchiseptica counts in nasal swabs before and after inoculation

[0081] Note: The unit is CFU / ml. “—” means no detection, and “ / ” means the test dog has been autopsied but not detected.

[0082] Table 4 Results of Bordetella bronchiseptica counts in different organs of dogs in each group

[0083] Note: The unit is CFU / g, “—” means not detected.

[0084] 2.4.2 Virulence reversion test

[0085] The viable bacterial content of Bordetella bronchiseptica HN60 strain culture was adjusted to 1×10 10CFU / ml, 2 dogs were inoculated intranasally, 2ml / dog, 7 days after inoculation, the 2 inoculated dogs were dissected, submandibular lymph nodes and tracheal lymph nodes were taken, weighed separately, added with PBS (0.01mol / L, pH7.2) at a ratio of 1:10 (w / v), ground with a grinder and filtered with an 80-mesh filter, and the filtrate of the ground submandibular lymph node of each dog was sampled for Bb live bacteria count, and the filtrate of the ground submandibular lymph node and organ lymph node of the 2 dogs was mixed and inoculated intranasally with another 2 blank dogs, 2ml / dog. According to the above method, the dog body was continuously passaged for 5 generations. If Bordetella bronchiseptica could not be re-isolated from the submandibular lymph node and tracheal lymph node during the passage process, the inoculation dose and the number of test animals should be doubled and the test should be repeated. The results showed that the canine bronchiseptica Bordetella HN60 strain was continuously propagated in dogs for two generations, and no abnormalities were found in the autopsy of the inoculated dogs. No bronchiseptica Bordetella was detected in the organs of the second-generation dogs. The results are shown in Table 5. The inoculation dose and the number of inoculated dogs were doubled and the test was repeated. The results showed that no bronchiseptica Bordetella was detected in the organs of the dogs in the second passage. The results are shown in Table 6. The above results show that the canine bronchiseptica Bordetella HN60 strain does not have a reversion to virulence.

[0086] Table 5 Results of Bb viable counts in different organs

[0087] Note: The unit is CFU / g, “—” means not detected.

[0088] Table 6 Results of Bb viable counts in different organs after doubling the inoculation dose and the number of inoculated dogs

[0089] Note: The unit is CFU / g, “—” means not detected.

[0090] 2.5 Horizontal transmission test of Bordetella bronchiseptica in dogs

[0091] The viable bacterial content of Bordetella bronchiseptica HN60 strain culture was adjusted to 1×10 10CFU / ml, 2 healthy susceptible dogs were inoculated with 2ml / dog by intranasal drops, and 2 blank dogs were transferred after inoculation and placed in the same cage with the inoculated dogs for co-rearing. Nasal swabs were collected from the dogs before co-rearing, 3 days, 7 days, 10 days, 14 days, and 21 days of co-rearing to detect the live bacteria content of Bordetella bronchiseptica in the nasal swabs; blood was collected before inoculation or cohabitation, 7 days, 14 days, and 21 days after inoculation or cohabitation to detect Bb microagglutination antibodies; on the 21st day after inoculation, all dogs were dissected and submandibular lymph nodes and tracheal lymph nodes were taken, and grinded separately for Bordetella bronchiseptica count. The results showed that Bordetella bronchiseptica could be detected in the inoculated dogs on the 3rd and 7th days, but Bordetella bronchiseptica was not detected on the 10th day and later after inoculation. Bordetella bronchiseptica was not detected in the nasal swabs of the cohabiting dogs from the 3rd to the 21st day of co-rearing. The specific results are shown in Table 7. The microagglutination antibodies of Bordetella bronchiseptica in the serum of dogs at different time points after vaccination or cohabitation were measured. The results showed that the Bordetella bronchiseptica antibodies of the vaccinated dogs were all lower than 1:20 before vaccination. 7-21 days after vaccination, a higher level of Bordetella bronchiseptica antibodies was produced, and the antibodies showed an upward trend. The Bordetella bronchiseptica antibodies of the cohabiting dogs were all lower than 1:20 before cohabitation and 7-14 days after cohabitation. The specific results are shown in Table 8. On the 21st day of vaccination or cohabitation, all dogs were autopsied. No abnormalities were found in the autopsies of all dogs. The submandibular lymph nodes and tracheal lymph nodes of each dog were taken for Bordetella bronchiseptica count. The results showed that Bordetella bronchiseptica were detected in both the submandibular lymph nodes and tracheal lymph nodes of the vaccinated dogs, while Bordetella bronchiseptica were not detected in the submandibular lymph nodes and tracheal lymph nodes of the two cohabiting dogs. The specific results are shown in Table 9. Based on the above results, there is no horizontal transmission after dogs are inoculated with Bordetella bronchiseptica HN60 strain.

[0092] Table 7 Results of Bordetella bronchiseptica counts in nasal swabs of dogs at different time points of inoculation or cohabitation (CFU / ml)

[0093] Note: “—” means not detected.

[0094] Table 8 Results of Bordetella bronchiseptica microagglutination antibodies in serum of dogs at different time points of inoculation or cohabitation

[0095] Table 9 Results of Bordetella bronchiseptica detection in different lymph nodes of dogs vaccinated or cohabiting for 21 days

[0096] Note: The unit is CFU / g, “—” means not detected

[0097] Example 3 Preparation of Bordetella bronchiseptica attenuated live vaccine

[0098] The attenuated strain HN60 of Bordetella bronchiseptica was cultured on a solid medium of Bo Jiang's blood plate, and a single colony was selected and cultured in a liquid medium containing BHI for 16 h. The colony was then transferred and expanded at a ratio of 1% until the viable bacterial concentration was not less than 5.0×10 9 CFU / mL. Add 20g sucrose and 2g gelatin to every 100ml deionized water, fully melt, and sterilize by high pressure steam (121℃30min). Add lyophilization protective agent at a ratio of 1:1 between bacterial liquid and protective agent (v / v), and freeze dry in a freeze dryer. The specific ratio of vaccine content is shown in Table 10.

[0099] Table 10 Content ratio of Bordetella bronchiseptica HN60 attenuated live vaccine Components Vaccine 1 Vaccine 2 Vaccine 3 HN60 antigen (CFU) / head <![CDATA[10 7.0 ]]> <![CDATA[10 8.0 ]]> <![CDATA[10 9.0 ]]> Protective agent (V / V) 50％ 50％ 50％

[0100] Example 4 Safety test of Bordetella bronchiseptica HN60 attenuated live vaccine

[0101] Fifteen healthy susceptible dogs about 60 days old were randomly divided into three groups, each with 5 dogs, and immunized with the attenuated live vaccine of Bordetella bronchiseptica HN60 prepared in Example 3. Group 1 was immunized with vaccine 1, Group 2 was immunized with vaccine 2, and Group 3 was immunized with vaccine 3. Ten doses were inoculated by nasal drops for each dog, and the dogs were observed for 14 days after inoculation. The clinical symptoms of the dogs were observed every day, mainly mental state, whether there were symptoms such as coughing, sneezing, nasal secretions, eye secretions, vomiting, etc., and the body temperature was measured every day. The specific results are shown in Table 11.

[0102] Table 11 Safety of Bordetella bronchiseptica HN60 attenuated live vaccine for dogs Group quantity Clinical symptoms 1 5 Normal body temperature, no clinical symptoms 2 5 Normal body temperature, no clinical symptoms 3 5 Normal body temperature, no clinical symptoms

[0103] The results showed that after the dogs were immunized with an overdose of the Bordetella bronchiseptica HN60 attenuated live vaccine prepared in Example 3, the body temperatures of the experimental dogs were normal and there were no clinical symptoms.

[0104] Fifteen healthy susceptible cats around 60 days old were randomly divided into 3 groups, 5 in each group, and immunized with the attenuated live vaccine of Bordetella bronchiseptica HN60 prepared in Example 3. Group 4 was immunized with vaccine 1, group 5 was immunized with vaccine 2, and group 6 was immunized with vaccine 3. Each cat was inoculated with 1 dose of intranasal drops, and observed for 14 days after inoculation. The clinical symptoms of the cats were observed every day, mainly mental state, whether there were symptoms such as coughing, sneezing, nasal secretions, eye secretions, vomiting, etc., and the body temperature was measured every day. The specific results are shown in Table 12.

[0105] Table 12 Safety of Bordetella bronchiseptica HN60 attenuated live vaccine for cats Group quantity Clinical symptoms 4 5 Normal body temperature, no clinical symptoms 5 5 Normal body temperature, no clinical symptoms 6 5 Normal body temperature, no clinical symptoms

[0106] The results showed that after cats were immunized with the attenuated live vaccine of Bordetella bronchiseptica HN60 prepared in Example 3, the body temperatures of all the experimental cats were normal and there were no clinical symptoms.

[0107] It has been proven that the Bordetella bronchiseptica HN60 attenuated live vaccine has good safety for both dogs and cats.

[0108] Example 5 Immunogenicity test of Bordetella bronchiseptica HN60 attenuated live vaccine in dogs

[0109] Twenty healthy susceptible dogs about 60 days old were randomly divided into 4 groups, 5 in each group, and immunized with the Bordetella bronchiseptica HN60 attenuated live vaccine prepared in Example 3. Group 7 was immunized with vaccine 1, group 8 was immunized with vaccine 2, and group 9 was immunized with vaccine 3. Each dog was immunized with one dose of intranasal drops, and the remaining group 10 was not vaccinated as a control. 28 days after vaccination, the immunized dogs and the control dogs were challenged with HN03 strain bacterial solution, 2 ml / dog (live bacteria content of 1.0×10 10 CFU / mL), observe continuously for 14 days after the virus challenge, record the clinical symptoms of each dog respectively, score the clinical symptoms according to the scoring criteria in Table 13, and perform statistical analysis on the average clinical symptom score of each group of dogs 14 days after the virus challenge. The average clinical symptom score of the immunized dogs should be significantly lower than that of the control dogs.

[0110] Table 13 Symptoms and scoring table of dogs after Bordetella infection

[0111] On the 28th day after vaccination, the immunized group and the control group were challenged with the virulent strain HN03, and the dogs were observed for 14 days after the challenge. The number of days and frequency of coughing and sneezing were recorded, and the scores were given according to the scoring table. The results showed that 5 / 5 dogs in the control group had symptoms of sneezing and coughing. The clinical symptom scores of the immunized groups with different doses of HN60 strain were significantly lower than those of the challenge control group, and all met the efficacy test standards. The specific results are shown in Table 14.

[0112] Table 14 Test dog immune challenge results

[0113] Note: “—” indicates no clinical symptoms, and different superscript letters indicate significant differences (P<0.05)

[0114] Example 6 Immunogenicity test of Bordetella bronchiseptica HN60 attenuated live vaccine on cats

[0115] Ten healthy susceptible cats around 60 days old were randomly divided into two groups, with 5 cats in each group. The Bordetella bronchiseptica live vaccine was diluted to 1.0×106 CFU / 0.5mL, the diluted vaccine was inoculated intranasally in 11 groups, 0.5ml per cat, and the 12th group was not inoculated as a control. On the 21st day after inoculation, the inoculated cats and the control cats were challenged intranasally with HN03 strain solution, 1ml / cat (live bacteria content of 1.0×10 9 CFU / mL), observe for 14 days after the infection, and record the clinical symptoms of each cat. At least 4 / 5 of the control cats should become ill (coughing for 2 consecutive days or more), and at least 4 immunized cats should be protected.

[0116] After 14 days of observation after infection, 5 / 5 cats in the control group became ill, while 5 / 5 cats in the immunized group were protected. The specific results are shown in Table 15.

[0117] Table 15 Results of cat immune challenge test

[0118] Note that the above are only preferred embodiments of the present invention and the technical principles used. Those skilled in the art will understand that the present invention is not limited to the specific embodiments described herein, and that various obvious changes, readjustments and substitutions can be made by those skilled in the art without departing from the scope of protection of the present invention. Therefore, although the present invention has been described in more detail through the above embodiments, the present invention is not limited to the above embodiments, and may also include more other equivalent embodiments without departing from the technical concept of the present invention, all of which belong to the protection scope of the present invention.

Claims

1. A Bordetella bronchiseptica attenuated strain HN60, It is characterized in that The accession number is CCTCCNO:M20221719, deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, and the deposit date is November 3, 2022.

2. Use of the Bordetella bronchiseptica attenuated strain HN60 or a culture thereof according to claim 1 in the preparation of a medicament for preventing Bordetella bronchiseptica infection.

3. The use according to claim 2, It is characterized in that The drug is a vaccine; Preferably, the subject is a dog or a cat.

4. A vaccine, It is characterized in that The vaccine comprises an immune effective amount of the Bordetella bronchiseptica attenuated strain HN60 or its culture according to claim 1, and pharmaceutically acceptable adjuvants.

5. The vaccine according to claim 4, It is characterized in that The content of Bordetella bronchiseptica attenuated strain HN60 is ≥1×10 6 CFU / head portion; preferably, the content of the attenuated strain HN60 of Bordetella bronchiseptica is 1×10 6 ~1×10 10 CFU / head portion; more preferably, the Bordetella bronchiseptica attenuated strain HN60 content is 1×10 9 CFU / head.

6. The vaccine according to claim 5, It is characterized in that The Bordetella bronchiseptica HN60 culture is a culture of the 1st to 60th generations.

7. The vaccine according to any one of claims 4 to 6, It is characterized in that The pharmaceutically acceptable excipient is a lyoprotectant, which includes SPGA, sugars, gelatin, protein, a substance containing protein or / and a buffer; Preferably, the sugar is selected from sorbitol, mannitol, starch, sucrose, glucose or dextran; the protein is selected from albumin or casein; the substance containing protein is bovine serum or skim milk; the buffer is phosphate buffer; Preferably, the lyoprotectant is 20w / v% sucrose and 2w / v% gelatin; Preferably, the volume ratio of the Bordetella bronchiseptica attenuated strain HN60 or its culture to the pharmaceutically acceptable excipient is 1:

1.

8. A method for preparing a vaccine according to any one of claims 4 to 7, It is characterized in that The Bordetella bronchiseptica attenuated strain HN60 or its culture is mixed with pharmaceutically acceptable adjuvants to obtain a vaccine.

9. A highly virulent strain of Bordetella bronchiseptica HN03, It is characterized in that The virulent strain HN03 of Bordetella bronchiseptica is homologous to the virulent strain HN60 of Bordetella bronchiseptica, and its preservation number is CCTCCNO:M20221718. It is preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, and the preservation date is November 3, 2022.

10. Use of the highly virulent Bordetella bronchiseptica strain HN03 according to claim 8 in testing the efficacy of a Bordetella bronchiseptica vaccine.

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