CN113801812A

CN113801812A - Pasteurella multocida and application thereof

Info

Publication number: CN113801812A
Application number: CN202111027185.1A
Authority: CN
Inventors: 郭爱珍; 袁永鹏; 郝成武; 贺笋; 韩瑞; 李庆妮; 张宇豪; 杨瑞琪; 陈颖钰; 胡长敏
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2021-09-02
Filing date: 2021-09-02
Publication date: 2021-12-17
Anticipated expiration: 2041-09-02
Also published as: CN113801812B

Abstract

The invention discloses a Pasteurella multocida (Pasteurella multocida) PmA-HG, which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2021805. The invention also discloses application of the strain in preparation of a bovine pasteurella multocida vaccine, belonging to the field of vaccine microorganisms. The invention screens out a vaccine-making strain with strong toxicity from a pasteurella multocida A type clinical isolate, then the vaccine-making strain is respectively matched with a plurality of adjuvants to screen out an adjuvant which can greatly improve the serum antibody level and the immune protective power, and finally the inactivated vaccine is prepared.

Description

Pasteurella multocida and application thereof

Technical Field

The invention belongs to the field of vaccine microorganisms, and relates to a pasteurella multocida vaccine strain and application of the strain.

Technical Field

Pasteurella multocida (Pm) usually colonizes the upper respiratory and gastrointestinal mucosa of animals and under stress conditions enhances adhesion to respiratory epithelial cells, causing severe respiratory and systemic diseases, resulting in huge economic losses. Pasteurella multocida comprises mainly A, B, D, E, F capsular serotypes, of which type a (pma) is the main serotype responsible for bovine respiratory disease syndrome. In recent years, the number of types F isolated from bovine tissues afflicted with respiratory disease in cattle tends to increase gradually. Although research and development on pasteurella multocida type A vaccines have been reported, the cross-protection effect between the pasteurella multocida type A vaccines and other serotypes (such as PmF type) is unknown, and the cross-immune effect on other serotypes is poor, so that the application of the vaccines in the control of bovine respiratory disease syndrome diseases is limited.

Meanwhile, most inactivated vaccines have weak ability to induce immune response, and rely on adjuvants to enhance the immune efficacy of the vaccine. The adjuvant can stimulate the immune response of organisms by slowly releasing antigens, stimulating antigen presentation reaction and the like, and is an important component of the vaccine. The clinical common adjuvants comprise various types such as oil emulsion adjuvants and water-based adjuvants, and the duration and side effects of the induced immunity are quite different. The oil adjuvant is difficult to be absorbed by tissues, and the economic value of muscles of meat animals such as beef cattle and the like is influenced; the water adjuvant is easy to absorb, but the antigen is released quickly, the retention time is short, and the side effect caused by the antigen is large.

Based on the reasons, the research screens out a vaccine-making strain with strong toxicity from separated and identified A-type clinical isolates of the Pasteurella multocida, then the vaccine-making strain is respectively matched with a plurality of adjuvants to screen out an adjuvant capable of greatly improving the serum antibody level and the immune protective power, and finally the inactivated vaccine is prepared.

Disclosure of Invention

One of the purposes of the invention is to provide a pasteurella multocida vaccine strain which has strong toxicity, can kill mice completely under 30CFU, and can provide better immune protection for animals, and simultaneously, the antibody produced by the strain in vivo has cross protection capability on A, F bovine pasteurella multocida serotypes, and can provide immune protection for A, F infection of the two serotypes.

The strain is classified and named as Pasteurella multocida PmA-HG, and has been preserved in China center for type culture Collection at 29 months 6 in 2021 with the preservation number of CCTCC NO: m2021805.

The invention also aims to provide a pasteurella multocida vaccine which is an inactivated vaccine and contains the strain and the immunologic adjuvant.

Preferably, the immunological adjuvant is Montanide GEL-02 water-soluble polymer adjuvant, and the result shows that the adjuvant can greatly improve the serum antibody level of animals after immunization, and simultaneously improve the survival rate of the animals after challenge, so that the vaccine has better immune protection.

It is a further object of the present invention to provide a method for preparing said bovine pasteurella multocida vaccine, comprising: culturing the strain with a TSA culture medium containing 4% of newborn calf serum to obtain a bacterial liquid, adding 0.2% of formaldehyde, inactivating at 37 ℃ for 18h, and adding an immunologic adjuvant after the bacterial liquid is qualified.

The cross protection test result shows that the vaccine provided by the invention can simultaneously provide immune protection for two serotypes of PmA and PmF, while the other PmF strong strain (PmF-XJ) inactivated vaccine can only provide immune protection for the serotype of PmF.

For a more detailed description, reference is made to the specific examples.

Drawings

FIG. 1: the influence of six immunologic adjuvants on the antibody production capacity of the PmA-HG inactivated vaccine.

Detailed Description

The present invention will be described in detail with reference to specific examples.

1 Material

1.1 strains

11 PmA clinical isolates and 3 PmF clinical isolates are both separated and identified from the upper respiratory tract or lung tissue of cattle with respiratory disease syndrome in the laboratory, and the strain information is listed in Table 1.

TABLE 1 information on the type A and type F strains of Pasteurella multocida used in this study

1.2 Experimental animals and ethical statements

18-22g healthy female Kunming mice (SPF grade), obtained from the animal laboratories center of university of agriculture in Huazhong. Animal experiments were approved by the ethical ethics committee of the university of agriculture in Huazhong (ethical number: HZAUMO-2021-.

1.3 Main Medium and reagents

Soybean trypsin Broth (TSB, Tryptic Soy Broth); tryptic Soy Agar (TSA, Tryptic Soy Agar); physiological saline; coating buffer solution; PBS buffer solution; wash solution (PBST); confining liquid (5% skim milk).

Types of adjuvants:

Imject^TMalum adjuvant was purchased from seemer feishell Scientific (Thermo Scientific) and was mainly composed of a suspension of aluminum hydroxide and magnesium hydroxide;

montanide GEL-02 is a water-soluble polymeric adjuvant from Sepeciaceae (SEPPIC) having as a major component a dispersion of high molecular weight polyacrylic acid in water;

ruienbei is purchased from Tangshan Yian bioengineering, Inc. and is a water adjuvant;

the propolis adjuvant is a gift from Shandong Binzhou animal husbandry research institute;

the Guhuhen investigator team of the Lanzhou veterinary institute of Chinese agricultural academy of sciences benefits self-made aqueous adjuvant, and is numbered as adjuvant 1;

the homemade veterinary nano adjuvant (sample No. 2) is a gift from the agricultural environment and sustainable development institute of the Chinese academy of agricultural sciences, and is numbered as adjuvant 2.

2 method

2.1 screening of candidate vaccine strains of Pma type

60 mice were divided into 12 groups, 5 mice in each group, 11 strains of Pma bacteria were intraperitoneally injected into each group 1-11, 30CFU in each group, and the same amount of physiological saline was injected into the 12 th group as a control, and appetite and mental status were observed every day after injection and death was recorded.

2.2 PmF-type virulence (LD) for mice₅₀) Measurement of (2)

Randomly dividing 50 SPF mice 18-22g into 10 groups, each group comprises 5 mice, respectively diluting 3 PmF bacteria solutions to 10CFU/mL, 1x10⁴CFU/mL，1x10⁷CFU/mL dilution of 9 groups, to the mouse peritoneal injection 200 u L; group 10 injections equal amounts of physiologySaline was used as a negative control. The clinical response of the mice was observed, the number of mouse deaths was recorded, and the LD of each strain was roughly calculated according to the modified Kouyveromyces method₅₀And screening out the strain with stronger toxicity as a candidate vaccine strain. Then reducing the attacking group distance of the virulent strains and calculating more accurate LD₅₀And (6) obtaining the result.

2.3 preparation of inactivated vaccines

2.3.1 preparation of bacterial liquid

Inoculating the candidate vaccine strain in TSA culture medium containing 4% newborn calf serum, culturing at 37 deg.C for 24 hr, selecting single colony, transferring, purifying and culturing for 1 time, selecting single colony, inoculating in TSB liquid culture medium containing 4% newborn calf serum, culturing at 37 deg.C for 200r min^-1Shake culturing for 16h to obtain seed liquid; according to the following steps: transferring the seed liquid into a TSB liquid culture medium containing 4% newborn calf serum according to the proportion of 100, and carrying out shaking culture at 37 ℃ for 12h to obtain a bacterial liquid.

2.3.2 inactivation of bacterial liquid

Adding formaldehyde solution into Pm bacterial solution at final concentration of 0.15%, 0.2%, 0.25% and 0.3%, respectively, placing in a constant temperature shaking incubator at 37 deg.C, and reacting at 180r/min for 12 hr, 18 hr and 24 hr, respectively. And respectively inoculating the inactivated bacterial liquid into a TSB liquid culture medium containing 4% of bovine serum and a TSA solid culture medium containing 5% of bovine serum, and culturing at 37 ℃ for 24h to check the inactivation effect.

2.3.3 preparation and testing of vaccines

And (4) completely inactivating the bacteria liquid, and adding an adjuvant according to the corresponding adjuvant use instruction to prepare the inactivated vaccine. Dropping a proper amount of the prepared adjuvant vaccine into clear water, and observing the diffusion condition in the water; and (3) centrifuging the mixture for 30min at 2500r by adopting an accelerated centrifugation method, and observing the dispersion condition of the adjuvant vaccine. The vaccines qualified in the test are respectively inoculated into SPF-grade 18-22g Kunming mice, and are injected into each vaccine at multiple points of 0.2mL per mouse, and each vaccine is inoculated with 10 mice. Two weeks of observation, mice health status was recorded and vaccine safety was evaluated.

2.4 Indirect ELISA for antibody titer detection

Overnight cultured Pm species were inoculated at a 1: 100 is inoculated in a TSB culture medium, cultured for 6h at 37 ℃ by 180 r.min < -1 > in a shaking table, then centrifuged at 12000r for 10min, the supernatant is discarded, the precipitate is resuspended by PBS with the same volume, and the resuspension solution is crushed by an ultrasonic crusher to prepare the holothurin. The BCA protein detection kit is used for detecting the protein concentration, the indirect ELISA reaction condition is optimized by a matrix titration method, and the antigen coating concentration and the serum dilution corresponding to the hole with the maximum positive OD630 nm/negative OD630nm (P/N value) are the optimal antigen coating concentration and the optimal antibody dilution.

2.5 screening experiments for pasteurella multocida vaccine adjuvants

Experiments compare the immunopotentiation effects of six adjuvants such as alum adjuvant, Ruienbibei water adjuvant, water-soluble polymer adjuvant (GEL 02), adjuvant 1 and adjuvant 2. Dividing 35 mice into 7 groups randomly, immunizing 6 PmA-HG strong strain adjuvant vaccines on the backs of 1-6 groups of mice respectively, injecting sterile PBS into the 7 th group as a control, carrying out secondary immunization by the same method after 14d, and injecting 10 times LD into the abdominal cavity of all mice after 14d of secondary immunization₅₀The PmA-HG strong strain is observed, the clinical symptoms, the morbidity and the mortality of the PmA-HG strong strain are observed and recorded, and dead mice are subjected to autopsy and bacteria isolation identification. The tail root vein blood collection and the serum separation are respectively carried out on each group of mice, and the antibody level in the serum of each group of mice is measured by an indirect ELISA method.

2.6 mouse Cross-protection test

Taking 40 healthy female SPF Kunming mice of 18-22g, dividing the mice into 2 groups, immunizing PmA-HG strain inactivated vaccine and PmF-XJ strain inactivated vaccine (the adjuvant uses water-soluble polymer adjuvant) respectively in each group, and immunizing neck and back under skin, wherein the method is the same as the previous method. And performing secondary immune 2w after the primary immune, and performing corresponding attack 2w after the secondary immune. Each group was subdivided into 3 groups and challenged with PmA-HG and PmF-XJ, respectively, at a dose of 10 XLD₅₀(ii) a An equal amount of saline was injected as an unstimulated control group. And observing for 14 days, and counting the protection rate of homologous strains and the cross protection rate of heterologous strains.

The protection rate (%) was calculated as follows:

the mice immune protection rate (%) — the number of surviving mice/total number of experimental mice × 100%.

2.7 statistical analysis

The experimental data were statistically analyzed by Graohpad prism 8.0, all expressed as mean. + -. standard deviation, with Student's T test for the comparison between experimental and control groups, Mann-Whitney U test for the comparison between two clinical analysis groups, and Kruskal-Wallis H test for the comparison between groups. Differences were considered statistically significant with p < 0.05.

3 results and analysis

3.1 screening of candidate strains of Pma vaccine

The results of the infection experiments of mice on 11 clinical isolates of Pma were all carried out at 30 CFU/mouse, and the result is shown in Table 2, wherein the death rate of the Pma-HG strain to the mice within 24h is 100%, and the strain shows the strongest toxicity.

TABLE 2 screening of pasteurella multocida type A virulence

3.2 PmF-type determination of virulence in mice (LD50)

The LD50, PmF-HN strain was roughly estimated to be 2.81X 10 according to the modified Kouzhi method⁴CFU/0.2mL, PmF-NY strain 5.01X 10³CFU/0.2mL, PmF-XJ strain 2.23X 10²CFU/0.2 mL. only, it was determined that Pm-XJ strain was slightly more virulent than Pm F-HN strain and Pm F-NY strain (Table 3). Reducing the group pitch of the preliminary experiment, and performing LD₅₀Calculating, the LD of the result PmF-XJ₅₀Is 2.18X 10²CFU/0.2 mL. only (Table 4).

TABLE 3 bovine pasteurella multocida type F isolate mouse virulence pretest

TABLE 4 LD50 determination of Pasteurella multocida PmF-XJ

3.3 preparation of inactivated vaccines

3.3.1 inactivation of bacterial liquid

As can be seen from Table 5, when formaldehyde was added to the bacterial suspension at a ratio of 0.2% (total volume of bacterial suspension), complete inactivation was achieved at 37 ℃ for 18 hours.

TABLE 5 inactivation test results of bacterial liquid

Note that + indicates that the test result is positive, + -indicates that the test result is suspicious, -indicates that the test result is negative.

3.3.2 preparation and testing of vaccines

Dropping alum adjuvant and adjuvant 2 on the surface of double distilled water, observing that the vaccine sinks to the water bottom or floats on the water surface, not diffusing, and slightly shaking the vaccine to disperse the vaccine into aggregated particles; the vaccine is centrifuged at 2500r/min for 30min, and the adjuvant 2 has no layering phenomenon, so that no emulsion breaking indicates that the emulsification effect is good; alum adjuvant has layering phenomenon, and should be injected after shaking before immunization.

Dropping a water-soluble polymer adjuvant (GEL 02), a Ruienbei adjuvant, a propolis adjuvant and an adjuvant 1 on the surface of double distilled water, and then rapidly fusing with water, wherein the vaccine adjuvant is a water-soluble adjuvant; the vaccine is centrifuged at 2500r/min for 30min without layering, which indicates that the emulsification effect is good.

The prepared vaccine is used for immunizing mice subcutaneously, and the living conditions of the mice are observed to be free from abnormality and subcutaneous necrosis and rot.

3.4 Indirect ELISA for antibody titer detection

The optimal coating concentration of holoprotein was determined to be 0.5. mu.g/mL, 100. mu.l/well, mouse antibody sera from 1: 200 start dilution, and the dilution multiple of the HRP goat anti-mouse secondary antibody is 1: 5000. This method was then used to compare the immunopotentiating effects of the adjuvants.

TABLE 6 test for optimal coating concentration of antigen

Antigen coating concentration (μ g/mL)	1	0.5	0.25	0.125	0.0625
						P	0.116	0.106	0.105	0.084	0.079
N	2.137	1.738	1.273	0.769	0.598
						N/P	18.42	16.55	12.01	9.15	7.57

TABLE 7 serum optimal dilution multiple test

Dilution factor	100	200	400	800	1600
						P	0.118	0.106	0.089	0.088	0.065
N	1.556	1.738	1.334	1.106	0.877
						N/P	13.18	16.55	15.16	12.42	13.50

3.5 screening of pasteurella multocida vaccine adjuvants

Alum (AI (OH))₃) The immunity enhancing effect of six adjuvants such as adjuvant group, adjuvant 1 group, adjuvant 2 group, propolis group, Ranuncut fritillary adjuvant group and water-soluble polymer adjuvant (GEL 02). The results showed that 14d after the priming (28 days after priming), the serum antibody level was highest in the group of water-soluble polymer adjuvant (GEL 02) and significantly higher than in the other group (p)<0.05). (FIG. 1)

The challenge results show that the alum adjuvant vaccine, the adjuvant 2 vaccine, the propolis adjuvant vaccine and the ranbexate water adjuvant vaccine all start to die after challenge, and the final die all occur on the 4 th day after challenge. The water soluble polymer adjuvant (GEL 02), adjuvant 1, adjuvant 2, alum, ranuncumber, propolis and non-adjuvant vaccine protection rates were 100%, 60%, 20% and 20%, respectively. Control mice all died within 24h after challenge (table 8). The experiment is combined to determine that the GEL 02 water-soluble polymer adjuvant is the optimal adjuvant of the PmA-HG inactivated vaccine, and can resist the 10LD of the strain by 100 percent after the mice are immunized₅₀Challenge with dose.

TABLE 8 Effect of five immunological adjuvants on the immunoprotective efficacy of vaccines

3.6 mouse Cross-protection test

After secondary immunization for 14 days, two serotype virulent strain attacks are respectively carried out on the PmA type and PmF type immunization groups, and the attacking dose is 10LD₅₀The results show thatThe immunity of the two bacteria can resist the attack of homologous strains by 100 percent; Pma-HG inactivated vaccine for immunizing mice with 10LD₅₀The cross protection rate of PmF attack is 100% (10/10), while the PmF-XJ inactivated vaccine immunized mice have 10LD₅₀The cross-protection rate of PmA challenge was only 10% (1/10) (Table 9), indicating that PmA-HG type strains could provide immune protection for both PmA and PmF serotypes.

TABLE 9 Cross-protective Effect of bovine Pasteurella multocida types A and F in mice

Claims

1. The Pasteurella multocida (Pasteurella multocida) PmA-HG is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2021805.

2. Use of a strain according to claim 1 for the preparation of a bovine pasteurella multocida vaccine, wherein antibodies raised in the animal are cross-protective against A, F two bovine pasteurella multocida serotypes, and are capable of providing immune protection against A, F two bovine pasteurella multocida serotypes simultaneously.

3. A pasteurella multocida vaccine which is an inactivated vaccine comprising the strain of claim 1 and an immunological adjuvant.

4. The bovine pasteurella multocida vaccine of claim 3, wherein: the immunological adjuvant is Montanide GEL-02 water-soluble polymer adjuvant.

5. A process for preparing the bovine pasteurella multocida vaccine of claim 3 or 4, characterized in that: the strain of claim 1 is cultured into a bacterial liquid by a TSA culture medium containing 4% of newborn bovine serum, then 0.2% of formaldehyde is added, inactivation is carried out for 18h at 37 ℃, and immune adjuvant is added after the test is qualified.