CN116350763A - Attenuated strain vaccine composition of bordetella bronchiseptica and preparation method thereof - Google Patents
Attenuated strain vaccine composition of bordetella bronchiseptica and preparation method thereof Download PDFInfo
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Abstract
The attenuated strain (YZ 01-P strain) of the canine bronchiseptica is genetically homologous to pathogenic bacteria of the canine bronchiseptica which are prevalent in China, and after animals are immunized by the vaccine composition prepared by the attenuated strain, the vaccine composition can induce animal organisms to quickly generate good mucosal immunity, the immunity efficacy can be maintained for a long time, and the occurrence of canine infectious respiratory diseases caused by the infection of the virulent strain of the canine bronchiseptica can be effectively prevented in the immunity efficacy maintenance period, and meanwhile, the occurrence of canine infectious diseases which are characterized by respiratory diseases and are caused by the mixed infection of the virulent strain of the canine bronchiseptica, canine distemper, canine parainfluenza and canine adenovirus can be reasonably reduced by .
Description
Technical Field
The invention relates to a canine bronchiseptica attenuated strain vaccine composition and a preparation method thereof, belonging to the technical field of veterinary biological products.
Technical Field
Infectious respiratory diseases caused by the infection of dogs with bordetella bronchiseptica (CanineBordetella Bronchiseptica, CBb). The dog bronchogenic bordetella belongs to the genus bordetella, and is gram-negative and aerobic. CBb was first isolated from canine respiratory tract with canine distemper by Ferry in 1910, and subsequently it was isolated from guinea pig, monkey and human respiratory tract in turn in 1912 and 1913 respectively (Ferry, n.s. a preliminary report ofthe bacterial findings in canine distemp. American Veterinary Review37:499-504.1010;Ferry,N.S.Further studies on the Bacillus bronchicanis,the cause of canine distemper.American Veterinary Review 41:77-79.1912).
Bordetella bronchiseptica (Bordetella Bronchiseptica, bb) and bordetella pertussis (Bordetella pertussis) and bordetella parapertussis (bordetella parapertussis) belong to a group, and are pathogenic bacteria which can infect various animals and cause respiratory tract related diseases. Bordetella bronchiseptica is pathogenic only to mammals and occasionally infects humans. Bb of porcine origin can co-infect with Pasteurella suis, resulting in atrophic rhinitis in pigs that is a serious health hazard to herds. Infection of rabbits with rabbit Bb can cause Rabbit Botrytis, which is extremely susceptible to outbreaks in autumn and winter where climate suddenly changes. The canine bronchogenic bordetella is an upper respiratory tract infection disease which is mainly characterized by cough and bronchopneumonia, and can be mixed with other viral pathogens of dogs, such as canine adenovirus, canine parainfluenza and canine distemper. CBb is highly pathogenic and rapidly transmitted, and severe cases can also cause death in infected dogs. The nasal secretions of animals with diseases, whether pigs, rabbits and dogs, contain a large amount of Bb pathogens, which can pollute feed, drinking water, cages and air with coughing and sneezing droplets, thereby infecting other susceptible animals.
In China, he Xingliang et al report on the separation of the upper respiratory tract of a cough-onset dog from the canine bronchogenic bordetella before and after 2002, and prove that the canine bronchogenic bordetella is widely popular in China and causes great harm to the health of dogs. Meanwhile, after sequencing and analyzing partial antigen gene sequences of CBb isolated bacteria, the canine bronchus-bordetella strain in China is considered to have 99 percent of similarity with a foreign reported reference strain, and also has 99 percent of homology with rabbit-source bronchus-bordetella strain sequences (He Xingliang. Separation and identification of canine bronchus-bordetella strain, livestock and veterinarian in 2004, volume 36, 12; snow white, separation and identification of canine bronchus-bordetella strain, cloning, expression and protein immunogenicity analysis of fim N genes. National university of Nanjing agricultural thesis, 2006).
According to foreign experience, the vaccine is a main measure for preventing and controlling the disease, but no commercial canine bronchogenic bordetella vaccine is sold and used in China at present. The publication number of the best company in 2006 filed in China a patent "canine vaccine against bordetella bronchiseptica", CN1835767a. The subunit vaccine is prepared by mixing a p68 recombinant protein antigen of the canine bronchiseptica with a veterinary acceptable carrier (such as an adjuvant), and the immune protection efficacy induced in dogs after injection cannot meet clinical requirements: after two vaccinations with 15 μg (the optimal dose in the above patent literature) of the p68 vaccine, the experimental dogs were given a virulent challenge on day 25 with only 20% protection (cough judged not to be protected for 2 consecutive days). Therefore, there is a great need to develop a canine bronchogenic bordetella attenuated live vaccine matched with the canine bronchogenic bordetella in China for preventing and controlling canine bronchogenic bordetella.
Disclosure of Invention
The invention aims to solve the defects of the prior art, and provides a canine bronchiseptica attenuated strain (YZ 01-P strain) which is matched with the antigenicity of Chinese epidemic bacteria and has good safety and immunogenicity, and a attenuated live vaccine prepared by using the attenuated strain, which are used for preventing and controlling canine infectious respiratory diseases caused by canine bronchiseptica infection.
The technical proposal of the invention
1. The attenuated live vaccine composition for the canine bronchiseptica bordetella disease is characterized by comprising attenuated bacteria YZ01-P strain matched with the antigenicity of the canine bronchiseptica bordetella which is popular in China;
the total genome sequence of the attenuated strain YZ01-P is 5339763bp, the total genome sequence codes 5039 genes, and the CG content of the genome is 68%. Compared with other disclosed sequences of the strain of the canine bronchiseptica bordetella, YZ01-P has two molecular genetic markers: (1) At 2955623-2955631 of the genome, there is an 8 base (CGCCGAGG) insertion; (2) there are 1 plasmid of 75606 bp;
the strain is delivered to China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, which is the national academy of sciences of China, no. 3 of China, which is the national academy of sciences of microorganisms, north China, which is the Korean area, as 2022, for 26 days, and the preservation number is: cgmccno. 2696.
2. The preparation process of the canine bronchogenic bordetella attenuated live vaccine comprises the following steps: culturing and proliferating attenuated bacteria, measuring antigen content, adding pharmaceutically acceptable carrier, sub-packaging, lyophilizing, and vacuum capping.
The positive effects of the invention
The attenuated strain (YZ 01-P strain) of the canine bronchiseptica is genetically homologous to pathogenic bacteria of the canine bronchiseptica which are prevalent in China, and after animals are immunized by the vaccine composition prepared by the attenuated strain, the vaccine composition can induce animal organisms to quickly generate good mucosal immunity, the immunity efficacy can be maintained for a long time, and the occurrence of canine infectious respiratory diseases caused by the infection of the virulent strain of the canine bronchiseptica can be effectively prevented in the immunity efficacy maintenance period, and meanwhile, the occurrence of canine infectious diseases which are characterized by respiratory diseases and are caused by the mixed infection of the virulent strain of the canine bronchiseptica, canine distemper, canine parainfluenza and canine adenovirus can be reasonably reduced by .
The invention relates to microbial resource information
The microorganism resource related in the invention is a canine bronchiseptica Botrytis attenuated strain YZ01-P, which is obtained by the inventor performing breeding, domestication and purification for at least 5 times from swab sample mixtures of various field pet dogs. The strain is delivered to China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, which is the national academy of sciences of China, no. 3 of China, which is the national academy of sciences of microorganisms, north China, which is the Korean area, as 2022, for 26 days, and the preservation number is: CGMCC No.26296.
Drawings
FIG. 1 shows PCR electrophoresis patterns of fimN gene of each generation of the attenuated bordetella bronchiseptica YZ01-P strain, wherein: m: DL2000 DNA markers; 1 to 23: YZ 01-P3-P25; n: negative control
FIG. 2 full Gene sequencing map of YZ01-P
FIG. 3 plasmid whole gene sequencing map of YZ01-P16
Detailed description of the invention
Domestication and breeding of YZ01-P strain
The microorganism resource related in the invention is a canine bronchiseptica Botrytis attenuated strain YZ01-P, which is obtained by the inventor performing breeding, domestication and purification for at least 5 times from swab sample mixtures of various field pet dogs. The acclimatization and breeding process is briefly described: and (3) collecting various swab and tissue organ samples of various pet dogs in animal hospitals, directly coating the samples (or preservation) on an improved MAIKAI culture dish, culturing in a 37 ℃ incubator for 36-48 hours, observing colonies with naked eyes, and selecting the colonies conforming to the growth form of the bordetella bronchiseptica to perform pure culture and identification to obtain a natural isolate named YZ01-O strain.
The YZ01-O strain is repeatedly domesticated, bred, separated and purified by monoclonal and identified for at least 5 times by using an improved MAIKAI culture medium and a culture method, and finally a culture which is uniform in colony morphology, moderate in colony size and in dew-like white colony is established and is named as YZ01-P strain. The YZ01-P strain is identified by gram staining and using the specific PCR of the dog bronchogenic bordetella, and is determined to be the dog bronchogenic bordetella; animal regression experiments were performed using YZ01-P strain, and YZ01-P strain was found to be a nonpathogenic attenuated bacterium. YZ01-P was grown in amplificatory culture and serial passage to passage 25 using conventional bacterial media, and its colony morphology, size, growth rate and FmN gene sequence all exhibited a stable state.
The whole genome sequence of the representative bacterium YZ01-P on the common bacterial culture medium is 5339763bp, the total genome sequence is 5039 genes, and the CG content of the genome is 68%. YZ01-P compared to isolates of other published sequences and to the full sequence of YZ01-O, YZ01-P has two molecular genetic markers: (1) At 2955623-2955631 of the genome, there is an 8 base (CGCCGAGG) insertion; (2) there are 1 75606bp plasmids.
2. Preparation of attenuated live vaccine
The vaccine related in the invention is a canine bronchogenic bordetella attenuated live vaccine, which is prepared by using canine bronchogenic bordetella attenuated strain (YZ 01-P strain) obtained by domestication, breeding and establishment of the inventor. The preparation process comprises the following steps: the method comprises the steps of carrying out recovery culture on the attenuated strain seeds of the bordetella bronchiseptica in a small-volume bacterial culture medium in a shaking table at 37 ℃ and 100-300 rpm for 8-24 hours, carrying out amplification culture according to 50-200 times, transferring the amplified culture into a 10-100L fermentation tank according to the proportion of 5-50 times, and carrying out high-density bacterial fermentation culture under the conditions of 37 ℃ and 100-500 rpm for 6-18 hours. After the bacterial antigen content of the fermentation culture solution is measured, the fermentation culture solution is mixed with a proper amount of freeze-drying protective agent, and then split charging is carried out, and finally freeze drying is carried out, thus obtaining the microbial inoculum.
3. Inspection of attenuated live vaccines
The safety and efficacy test of experimental dogs is carried out on the canine bronchogenic bordetella attenuated live vaccine related to the invention.
(1) Safety inspection of vaccine: 5 experimental dogs were vaccinated with 10-fold doses of attenuated live vaccine, 5 control groups were vaccinated with PBS, and all vaccinated animals were not abnormally reacted with the control group animals for 14 days. Proved to be safe.
(2) Efficacy test of vaccine: 10 test animals were vaccinated with 1-dose live attenuated vaccine, and 10 control animals were vaccinated with PBS. After 14 days, the animals of the immunized group and the animals of the control group are subjected to the virulent attack of the canine bronchiseptica, and the result is that 10 dogs of the test group are protected from any clinical symptoms including respiratory symptoms after 14 days, and 100% of animals of the control group have respiratory symptoms. Proved to be effective by the attenuated live vaccine.
The following description will be given of terms involved in the present invention
1. The term "bordetella bronchiseptica" (CBb) belongs to the genus bordetella. The clinical symptoms that are triggered include respiratory diseases and death characterized by cough. Clinically, it can be mixed with canine distemper, canine parainfluenza and canine adenovirus to cause more complex animal morbidity and serious clinical symptoms including animal death.
2. Compared with YZ01-O strain and other CBb strain with complete gene sequence determination and disclosure, the canine bronchiseptica attenuated bacteria (YZ 01-P strain) has two molecular genetic markers: (1) At 2955623-2955631 of the genome, there is an 8 base (CGCCGAGG) insertion; (2) there are 1 75606bp plasmids.
3. The method for separating the canine bronchiseptica bordetella comprises the following steps: taking animal organs, tissues and samples infected with bordetella bronchiseptica for bacterial isolation;
4. animal organs, tissues and samples infected with canine bordetella bronchiseptica of the present invention include, but are not limited to, trachea, lung, nasal secretions and the like.
5. The term "pharmaceutically acceptable carrier" refers to all components in the vaccine composition of the present invention except for the canine bordetella bronchiseptica YZ01-P strain antigen, which are mainly lyoprotectants: dipotassium hydrogen phosphate, potassium dihydrogen phosphate, casein hydrolysate, sorbitol, skimmed milk powder, hydrolyzed milk protein, gelatin, sucrose, lactose, and arginine.
6. The term "preventing and/or treating" when referring to a virulent strain of bordetella bronchiseptica refers to inhibiting replication of a virulent strain of bordetella bronchiseptica, inhibiting transmission of a virulent strain of bordetella bronchiseptica or preventing colonization and propagation of a virulent strain of bordetella bronchiseptica in its host, and reducing disease or symptoms in dogs caused by infection with a virulent strain of bordetella bronchiseptica.
Examples
The advantages and features of the present invention will become more apparent from the following description of the invention, taken in conjunction with the accompanying examples. These examples are merely exemplary and do not limit the scope of the invention in any way.
Example 1
Domestication, breeding and establishment of canine bronchiseptica attenuated bordetella YZ01-P strain
1. Isolation, purification and identification of the bordetella bronchiseptica YZ01-O strain
And (3) collecting samples of various swabs and tissues and organs of various pet dogs in animal hospitals, and rapidly inserting the collected samples into a sterile freezing tube containing preservation solution for preservation, or directly coating the samples on an improved Maiconk agar culture medium plate.
The preserved sample is taken and coated on a modified Maiconkai agar culture medium plate, and is cultivated for 36 to 48 hours in a constant temperature incubator at 37 ℃ for observation. Pure culture of single colonies of suspected bordetella bronchiseptica: single colonies were picked and streaked onto modified MAIKAI agar plates in pure culture, after 48 hours of incubation at 37℃the colony morphology was observed and colony PCR identified. For individual colonies identified as B.canis, a sterile spot picking operation was performed with an inoculating loop, inoculated into liquid medium for expansion culture (in tubes containing 5mL of medium), and inoculated again with an inoculating loop onto a modified MAIKAI agar medium plate for culturing at 37℃for 48 hours. The original isolate of the canine bronchogenic bordetella is obtained through gram staining and canine bronchogenic bordetella specific PCR identification, and is named YZ01-O.
2. Domestication, breeding and establishment of bordetella bronchiseptica YZ01-P strain
The improved Maikang Kai selective culture medium and the culture method are used for repeatedly domesticating, breeding, purifying and identifying YZ01-O for at least 5 times to obtain a white colony culture with uniform colony morphology and moderate colony size in dew-like shape. The strain is systematically identified by gram staining and using canine bronchogenic bordetella-specific PCR, and is determined to be canine bronchogenic bordetella; when the strain was passaged for 25 successive generations using a common medium, the colony morphology and size, bacterial growth rate, gram stain and FmN gene sequencing were all in a stable state for each generation (see FIG. 1). Animal regression experiments were further performed using this strain: use 10 9 PFU was not pathogenic to dogs for about 16 weeks of age, and CBb antibodies and antigen negative dogs were observed for 14 days by respiratory inoculation without other respiratory symptoms such as cough, and therefore the strain was considered to be a nonpathogenic attenuated strain for dogs. The establishment of a genetically stable strain of the attenuated strain of the canine bronchiseptica is confirmed, the strain is named YZ01-P strain, and the strain liquid can be stored at the temperature of minus 70 ℃ for a long time after being mixed with 5 to 20 percent of glycerol.
3. Identification of each generation of serial passages of the bordetella bronchiseptica YZ01-P strain
(1) Gram staining identification: and 5 mu L of bacteria liquid to be detected is dripped on the clean glass slide, and the clean glass slide is uniformly smeared. The slide with the bacterial film faces upwards and is rapidly rocked back and forth above the flame of the alcohol lamp for 3-5 times for fixation. And (3) dropwise adding crystal violet to the bacterial film for dyeing for 1min, washing, airing, adding iodine solution to cover the surface for dyeing for about 1min, washing, absorbing water by using absorbent paper, and drying. Adding 95% alcohol, slightly shaking for decolorizing, observing bacterial film on glass slide under white background, removing colorless or non-fading, washing with water, absorbing water with absorbent paper, and drying. Counterstaining: after counterstaining for 1min, the red dye solution was washed with water and dried by absorbing water. The cedar oil was added dropwise, and as a result, scattered gram-negative bacilli were observed by observation with an oil lens.
(2) Specific PCR and sequencing identification reference methods (isolation and identification of Brucella bronchiseptica and cloning, expression and expressed protein immunogenicity analysis of fim N gene. Nanjing university of agriculture, ind. 2006) A Brucella bronchiseptica specific PCR method was established, and the identification such as amplification and sequencing of fimN gene was performed on P3-to P25-generation bacterial cultures. (Each of the components of Table 1 below was added to a 0.2mL PCR reaction tube in a total reaction volume of 25. Mu.L, and a blank control was established except for the template to be examined.
The primer sequences are as follows:
FimN-F: gggaattcac gtgatcaccg 20 (sequence 1)
FimN-R: ggctcgagga ttatccttat caagcc 26 (sequence 2)
TABLE 1fimN reaction System
PCR procedure: the system is evenly mixed and then subjected to PCR amplification, and after being pre-denatured for 3min at 95 ℃, the following cycle is carried out: denaturation at 95℃for 30s, annealing at 56℃for 30s; the extension was carried out at 72℃for 40s for a total of 35 cycles. The mixture is subjected to a reaction at 72 ℃ for 10min and stored at 4 ℃.
After 1.5% agarose gel electrophoresis identification, the positive PCR product was commissioned to sequencing by Suzhou Hongsu biotechnology limited. Primers and reaction conditions for amplification are attached). FIG. 1 is a chart showing PCR electrophoresis of the fim N gene of isolated Brucella bronchiseptica YZ01-P3 to P25. Sequencing results are shown in sequence 3 and sequence 4, sequencing results are analyzed to prove that the homology of fimN genes of P3 and P25 is 100%, and the results prove that the canine bronchiseptica attenuated bacteria with stable genetic characters are established.
Sequencing results of fimN genes of Brucella bronchiseptica YZ01-P3 (SEQ ID NO: 3) and P25 (SEQ ID NO: 4).
YZ01-P3 (sequence 3):
YZ01-P25 (sequence 4):
(3) Whole genome sequencing analysis
Performing whole genome sequencing on the 16 th generation culture (YZ 01-P16) of the YZ01-P by adopting a second generation sequencing method, wherein the result is that the whole genome sequence of the YZ01-P16 is 5339763bp, and the GC content of the whole genome is 68.71%; wherein 90.42% of the whole genome is a gene coding region sequence, the total length is 4828254bp, the total length codes 5039 genes, and the average gene length is 958bp; the total length of the sequences between genes is 511509bp, the total length of the temporary genes is 9.58%, and the GC content is 62.13%. The whole gene map is shown in figure 2.
Comparing the obtained whole sequence of YZ01-P16 with the whole genome sequence of other dog bordetella bronchiseptica published by original isolate YZ01-O and NCBI, the obtained whole sequence of YZ01-P16 is subjected to genome analysis, and two molecular genetic markers are found: (1) At 2955623-2955631 of the genome, there is an 8 base (CGCCGAGG) insertion; (2) there are 1 75606bp plasmids (see FIG. 3).
(4) Content of live bacterial antigen in culture of each generation of bordetella bronchiseptica YZ01-P
The viable bacteria count method (Chinese animal pharmacopoeia 2020 edition, appendix 3405) is adopted to measure the viable bacteria content in each generation of YZ01-P culture, and the viable bacteria number in each generation of YZ01-P culture can reach 1×10 9 CFU/mL。
Example 2
Preparation of attenuated live vaccine of bordetella bronchiseptica strain
1mL of YZ01-P seeds are taken and are subjected to resuscitating culture for 8 to 24 hours in a shaking table with a small volume of bacterial culture medium at a temperature of between 37 ℃ and 100 and 300rpm, and then are subjected to amplified culture according to a ratio of 50 to 200 timesThen transferring into 10-100L fermenter according to 5-50 times ratio, fermenting and culturing at 37deg.C and 100-500 rpm for 6-18 hr, OD 600 The range is generally between 2 and 10. After the bacterial content of the fermentation liquor is measured, the fermentation liquor is diluted according to the formula to obtain bacterial solutions with different bacterial contents, the bacterial solutions are mixed with a freeze-drying protective agent, and then the mixed solution is packaged into penicillin bottles, and after the freeze-drying treatment is rapidly carried out, the sample is subjected to vacuum capping to obtain the vaccine composition. The vaccine composition obtained after lyophilization was subjected to content and purity tests (methods refer to appendixes 3405 and 3306 of the chinese pharmacopoeia 2020). The results prove that the vaccine composition has acceptable antigen content (not less than 10 6.5 CFU/ml/head), and has good purity and no other mixed bacteria pollution. The inventors continuously prepared 5 to 10 batches of experimental articles, selected for their high dose (10 9.5 A safety check was performed on a batch of CFU/ml/head, and medium dose (10) was selected 7.4 CFU/ml/head) a lot of the preparation was tested for efficacy.
Example 3
Inspection of attenuated live vaccine of bordetella bronchiseptica
1. Safety test of attenuated live vaccine against bordetella bronchiseptica
The safety test of single dose and 10 times dose overdose inoculation adopts about 2 months old, 15 healthy susceptible dogs are randomly divided into 3 groups and 5 groups, wherein 1mL of canine bronchogenic bordetella attenuated vaccine is inoculated in group 1, and the live bacteria content is 10 8.5 CFU per dog; group 2 was overdose vaccinated with 10-fold dose, vaccinated with 4mL of Botrytis cinerea attenuated vaccine with live bacteria content of 10 9.5 CFU per dog; group 3 was a placebo group vaccinated with 1ml fbs per canine. Feeding under the same condition after inoculation, observing for 14 days, and observing whether the dogs drink water and appetite, body temperature, eyes and noses are normal, whether adverse reactions exist on the whole body or not, and whether cough or other respiratory symptoms exist in clinical manifestations or not every day. The test dogs in the single dose and 10-times dose test group and the control group, which had good mental status, normal water and appetite, normal body temperature and no ocular and nasal secretion product, had the end of the observation periodRaw dogs showed healthy activity without cough and other respiratory symptoms, no adverse reaction to the whole body (results are shown in table 2).
TABLE 2 safety test results of canine bronchogenic bordetella attenuated live vaccine against one single and one overdose vaccination of dogs
Experimental results indicate that live vaccine of the bordetella bronchiseptica is safe for dogs of 2 months of age.
2. Efficacy test of live bordetella bronchiseptica vaccine
The method comprises the steps of randomly dividing 20 healthy susceptible dogs into 2 groups and 10 healthy susceptible dogs by about 2 months of age, wherein 1mL of live vaccine of the bordetella bronchiseptica is inoculated in the group 1, and the live bacterial content of the vaccine is 10 7.4 CFU/mL. Group 2 was a blank control, inoculated with 1ml fbs. After inoculation, the dogs are fed under the same condition and observed for 14 days, and all dogs tested are found to be normal in drinking water, appetite, body temperature and eyes and noses, have no adverse reaction on the whole body and have no cough or other respiratory symptoms in clinical manifestations. On day 14 after immunization, all test dogs are challenged by the virulent strain of the bordetella bronchiseptica, and after the challenge, whether drinking water, appetite, body temperature, eyes and noses of all the test dogs are normal or not is observed every day, adverse reactions exist on the whole body, and the clinical manifestations are no cough or death. The results are shown in Table 3.
TABLE 3 results of efficacy test of attenuated live vaccine against B.bronchiseptica
| Grouping | Number of animals | Dose of inoculation | Number of incidences | Number of deaths | |
| Vaccine | |||||
| 10 | 1 ml/1 ml only | 0/10 | 0/10 | 100％ | |
| Blank control (PBS) | 10 | 1 ml/1 ml only | 10/10 | 0/10 | 0％ |
The results show that the live vaccine of the bordetella bronchiseptica can provide 100% protection for dogs 14 days after the live vaccine of the bordetella bronchiseptica is used for immunizing healthy susceptible dogs about 2 months old respectively and the virulent strain of the bordetella bronchiseptica is used for attacking the live vaccine of the bordetella bronchiseptica. The vaccine can induce animals to generate good immunity protection and has good protection effect on virulent attack.
As can be seen from tables 2 and 3: the method is safe and effective for the canine bronchogenic bordetella attenuated live vaccine (YZ 01-P strain) to inoculate the healthy and susceptible dogs about 2 months old.
The present invention is not limited to the above-described preferred embodiments, but is intended to be limited to the above-described preferred embodiments, and any simple modification, equivalent changes and modification made to the above-described embodiments according to the technical matters of the present invention without departing from the scope of the present invention.
Claims (2)
1. A canine bronchogenic bordetella attenuated live vaccine composition is characterized in that the vaccine composition contains attenuated bacteria YZ01-P strain matched with the antigenicity of the canine bronchogenic bordetella epidemic in China;
the total genome sequence of the attenuated strain is 5339763bp, and the total genome sequence codes 5039 genes, and the CG content of the genome is 68%. Compared with other disclosed sequences of the strain of the canine bronchiseptica bordetella, YZ01-P has two molecular genetic markers: (1) At 2955623-2955631 of the genome, there is an 8 base (CGCCGAGG) insertion; (2) there are 1 plasmid of 75606 bp; the strain is delivered to China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, which is the national academy of sciences of China, no. 3 of China, which is the national academy of sciences of microorganisms, north China, which is the Korean area, as 2022, for 26 days, and the preservation number is: cgmccno. 2696.
2. The preparation method of the attenuated live vaccine of the bordetella bronchiseptica according to claim 1, wherein the preparation process comprises the steps of culturing and proliferating attenuated bacteria, measuring the antigen content, adding a pharmaceutically acceptable carrier, sub-packaging, freeze-drying and vacuum capping.
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