A kind of avian Escherichia coli O145 serotype inactivated vaccine and preparation method thereof

Abstract

The invention provides an avian Escherichia coli O145 serotype inactivated vaccine and a preparation method thereof. The avian Escherichia coli of the invention is Escherichia coli JX164, with a deposit number of CCTCC NO: M 20231988, which can be used to prepare an avian Escherichia coli O145 serotype inactivated vaccine; the avian Escherichia coli O145 serotype inactivated vaccine of the invention, after standing for 12 hours, the vaccine is evenly distributed without stratification; within 7 days after immunization, no obvious side effects occur in each group of ducks, the survival rate reaches 100% 7 days after immunization, and the vaccine has good safety; the avian Escherichia coli O145 type inactivated vaccine prepared by the invention can resist infection with the same serotype strong strain, provide a higher protection rate, but a lower cross protection rate.

Description

Technical Field

[0001] The invention relates to the technical field of inactivated vaccines, in particular to an inactivated vaccine of avian Escherichia coli O145 serotype and a preparation method thereof. Background Art

[0002] Avian colibacillosis is a systemic or local infection of poultry caused by avian pathogenic Escherichia coli (APEC). Clinically, it generally manifests as sepsis, omphalitis, salpingitis, peritonitis, synovitis, cellulitis, ascites, sinusitis and other symptoms, with high morbidity and mortality, causing huge losses to the poultry industry. Since plasmids carrying drug-resistant genes and virulence factors in APEC can be transmitted to other animal and human bacteria through the environment or food chain, they pose a threat to the health of humans and other animals. Therefore, APEC also has important public health significance.

[0003] For a long time, among the many serotypes of APEC, O1, O2 and O78 are considered to be highly related to poultry pathogenicity and are considered to be the three most important serotypes. However, recently, scholars from Nanjing Agricultural University proposed that the O145 serotype accounted for 37.9% (85 / 224) of the total number of strains in the epidemic survey, and its virulence and drug resistance were higher than the three traditional serogroups in my country. This result is highly consistent with the conclusion of the epidemic survey of the present invention. The epidemic survey data of the present invention show that the O145 serotype accounts for 16.6% (27 / 163) of the total number of strains in the epidemic survey. Compared with other serotypes, the O145 serotype is in a dominant position in clinical prevalence.

[0004] The complex serotypes of E. coli disease and the increasingly serious problem of drug resistance of avian E. coli have brought great challenges to the prevention and control of the disease. At present, there is no commercial vaccine for avian E. coli disease against the O145 serotype at home and abroad. Therefore, the development of an inactivated vaccine for avian E. coli disease of the O145 serotype is a new technology reserve for the prevention and control of E. coli disease, which can provide effective protection for the healthy development of my country's poultry industry. Summary of the invention

[0005] The invention provides an avian Escherichia coli O145 serotype inactivated vaccine and a preparation method thereof, so as to solve the defects existing in the prior art.

[0006] In a first aspect, the present invention provides an avian Escherichia coli, which is Escherichia coli JX164, with a deposit number of CCTCC NO: M 20231988.

[0007] In a second aspect, the present invention also provides a use of the avian Escherichia coli in preparing an inactivated vaccine of avian Escherichia coli O145 serotype.

[0008] In a third aspect, the present invention also provides an inactivated vaccine of avian Escherichia coli O145 serotype, comprising the avian Escherichia coli.

[0009] Preferably, in the inactivated vaccine of avian Escherichia coli O145 serotype, the antigen content of the inactivated Escherichia coli strain is ≥ 6.0×10 9 CFU / mL.

[0010] Preferably, the inactivated vaccine of avian Escherichia coli O145 serotype further comprises an adjuvant.

[0011] Preferably, the inactivated vaccine of avian Escherichia coli O145 serotype is prepared with aluminum gel adjuvant or MONTANIDEGEL P PR adjuvant.

[0012] Preferably, in the inactivated vaccine of avian Escherichia coli O145 serotype, the volume fraction of the adjuvant in the inactivated vaccine of avian Escherichia coli O145 serotype is 20-30%.

[0013] In a fourth aspect, the present invention further provides a method for preparing the avian Escherichia coli O145 serotype inactivated vaccine, which comprises the following steps:

[0014] Preparation of inactivated antigen of avian Escherichia coli strain: inoculating the Escherichia coli strain into LB medium for culture, and performing primary and secondary seed propagation respectively; then transferring culture and performing inactivation treatment;

[0015] Preparation of vaccine: dilute or concentrate the inactivated bacterial solution to make the number of live bacteria before inactivation ≥ 6.0 × 10 9 CFU / mL; take the inactivated bacterial solution and the adjuvant, mix them evenly, stir and emulsify them to obtain the avian Escherichia coli O145 serotype inactivated vaccine.

[0016] Preferably, in the method for preparing the inactivated vaccine of avian Escherichia coli O145 serotype, the conditions for the transfer culture are: the bacterial liquid of the secondary seed is transferred to a shaking bottle at a ratio of 1: (80-120), and the bacteria are cultured at 170-190 r / min in a shaking incubator at 35-38°C. After culturing to the plateau phase, the bacteria are centrifuged at 7000-9000 r / min in a high-speed refrigerated centrifuge for 2-4 min, the supernatant is discarded, the bacteria are collected, the bacteria are resuspended in physiological saline, 90-110 μL of the bacterial liquid is taken for multiple dilution, and the live bacteria concentration of the resuspended bacterial liquid is calculated by counting the live bacteria.

[0017] Preferably, in the method for preparing the inactivated vaccine of avian Escherichia coli O145 serotype, the inactivation treatment is specifically as follows: adding a formaldehyde solution with a volume ratio of 0.3-0.7% to the bacterial solution, and inactivating the solution at 35-38°C in a shaking incubator at 80-120 r / min for 40-60 hours.

[0018] The avian Escherichia coli O145 serotype inactivated vaccine and its preparation method have the following beneficial effects compared with the prior art:

[0019] The avian Escherichia coli of the present invention is Escherichia coli JX164, with a deposit number of CCTCC M 20231988, which can be used to prepare an inactivated vaccine of avian Escherichia coli O145 serotype; after standing for 12 hours, the vaccine of the inactivated vaccine of avian Escherichia coli O145 serotype is evenly distributed without stratification; within 7 days after immunization, no obvious side effects occur in each group of ducks, and the survival rate reaches 100% 7 days after immunization, and the vaccine has good safety; the inactivated vaccine of avian Escherichia coli O145 prepared by the present invention can resist infection by strong strains of the same serotype, provide a higher protection rate, but a lower cross-protection rate. Containing 20% ​​MONTANIDE TM The immune protection effect of the vaccine prepared with GEL P PR adjuvant is better than that of the vaccine prepared with 30% aluminum hydroxide gel adjuvant. DETAILED DESCRIPTION

[0020] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not 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.

[0021] The following are described in detail. It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of the embodiments. In addition, in the description of the present application, the term "including" means "including but not limited to". Various embodiments of the present invention may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and simplicity, and should not be understood as a rigid limitation on the scope of the present invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single values ​​within the range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the numbered ranges, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated in this article, it is meant to include any quoted numbers (fractions or integers) within the indicated range.

[0022] The invention provides an avian Escherichia coli, which is Escherichia coli JX164, and the preservation number is CCTCC NO: M 20231988.

[0023] The avian Escherichia coli (Escherichia coli) JX164 of the present invention is deposited in the China Center for Type Culture Collection, the deposit address is: Wuhan University, Wuhan, China, the deposit number is CCTCC NO: M 20231988, and the deposit date is October 23, 2023.

[0024] Based on the same inventive concept, the present invention also provides a use of the above-mentioned avian Escherichia coli in the preparation of an avian Escherichia coli O145 serotype inactivated vaccine.

[0025] Based on the same inventive concept, the present invention also provides an inactivated vaccine of avian Escherichia coli O145 serotype, comprising the above-mentioned avian Escherichia coli.

[0026] In some embodiments, the antigen content of the inactivated Escherichia coli strain in the avian Escherichia coli serotype O145 inactivated vaccine is ≥ 6.0×10 9 CFU / mL.

[0027] In some embodiments, an adjuvant is also included.

[0028] In some embodiments, the adjuvant is aluminum gel adjuvant or MONTANIDE GEL P PR adjuvant. Specifically, the aluminum gel adjuvant is aluminum hydroxide gel adjuvant.

[0029] In some embodiments, the volume fraction of the adjuvant in the avian Escherichia coli O145 serotype inactivated vaccine is 20-30%, that is, the volume ratio of the Escherichia coli strain to the adjuvant in the avian Escherichia coli O145 serotype inactivated vaccine is (80-70):(20-30).

[0030] Based on the same inventive concept, the present invention also provides a method for preparing the above-mentioned avian Escherichia coli O145 serotype inactivated vaccine, which comprises the following steps:

[0031] Preparation of inactivated antigen of avian Escherichia coli strain: inoculating the Escherichia coli strain into LB medium for culture, and performing primary and secondary seed propagation respectively; then transferring culture and performing inactivation treatment;

[0032] Preparation of vaccine: dilute or concentrate the inactivated bacterial solution to make the number of live bacteria before inactivation ≥ 6.0 × 10 9 CFU / mL; take the inactivated bacterial solution and the adjuvant, mix them evenly, stir and emulsify them to obtain the avian Escherichia coli O145 serotype inactivated vaccine.

[0033] In some embodiments, the conditions for transfer culture are: the bacterial liquid of the secondary seed is transferred to a shaking flask at a ratio of 1: (80-120), and the bacteria are cultured at 170-190 r / min in a shaking incubator at 35-38°C. After culture to the plateau phase, the bacteria are centrifuged at 7000-9000 r / min in a high-speed refrigerated centrifuge for 2-4 minutes, the supernatant is discarded, the bacteria are collected, and physiological saline is used to resuspend the bacteria. 90-110 μL of the bacterial liquid is taken for multiple dilution, and the viable bacteria concentration of the resuspended bacterial liquid is calculated by counting the viable bacteria.

[0034] In some embodiments, the inactivation treatment is specifically as follows: adding a 0.3-0.7% formaldehyde solution by volume to the bacterial solution, and inactivating the bacterial solution at 35-38° C. in a shaking incubator at 80-120 r / min for 40-60 h.

[0035] The following further describes the avian Escherichia coli O145 serotype inactivated vaccine and its preparation method of the present application with specific examples. This section further describes the content of the present invention in conjunction with specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0036] In the following examples, aluminum hydroxide gel adjuvant: aluminum hydroxide is a crystalline aluminum hydroxide, which is positively charged at physiological pH (pI=11) and suitable for adsorbing negatively charged acidic proteins (such as albumin). It is a commercial product purchased; MONTANIDE TM GEL P PR adjuvant is a newly developed polymer adjuvant, which is high molecular weight polypropylene and is a purchased commercial product; duck immunoglobulin G (IgG) enzyme-linked immunosorbent assay kit is a purchased commercial product.

[0037] Example 1: Collection and screening of vaccine strains

[0038] From April 2020 to December 2022, suspected avian Escherichia coli disease samples were collected from poultry farms in different regions of China. The inventors cut tissue blocks and soaked them in a 75% ethanol solution for 1 minute to remove pathogenic microorganisms on the surface of the liver; the tissue blocks were placed in sterile EP tubes and washed twice with 1.5 mL of sterile saline; the ground tissue blocks and aseptically collected liquid samples were spread on MacConkey plates and incubated at 37°C for 24 hours; single colonies with a dark pink center, round, flat, neat edges, smooth surface, and moist were streaked on LB plates and incubated at 37°C for 24 hours. The colonies were resuspended with sterile saline, one portion of the bacterial suspension was added with 25% volume of glycerol and stored in a -20°C refrigerator, and the other was sent to Shanghai Shenggong Biotechnology Co., Ltd. for whole genome sequencing. The sequencing results were analyzed online for serotype and virulence genes at the Center for Genomic Epidemiology.

[0039] Experimental results: 163 strains of avian pathogenic Escherichia coli were isolated and identified in the laboratory. The dominant serotype O145 was screened out through whole genome sequencing, serotype comparison, virulence gene analysis and animal pathogenicity tests.

[0040] After determining the dominant serotype according to the results of the Center for Genomic Epidemiology analysis, two strains with the largest number of virulence genes were selected for animal pathogenicity experiments. 80 7-day-old ducks were selected and divided into 8 groups, with 10 ducks per group. Four infection concentration gradients were set for each strain, namely 2.0×10 6 CFU / feather, 2.0×10 7 CFU / feather, 2.0×10 8 CFU / feather, 2.0×10 9 CFU / feather, artificial infection was carried out by intramuscular injection and observed continuously for one week. Strong strains were screened according to the pathogenicity judgment criteria of strains. The results are shown in Table 1 below.

[0041] Table 1 - Results of strain pathogenicity test

[0042]

[0043] As shown in Table 1, a highly virulent strain APEC-164 was obtained and named as Escherichia coli JX164, and deposited in China Center for Type Culture Collection, with the deposit address being: Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M 20231988.

[0044] Example 2: Screening of the optimal bacterial content of the vaccine

[0045] Take the frozen avian pathogenic Escherichia coli JX164 and streak it on the plate, place it in an incubator at 37°C and culture it overnight to recover. The next day, select single colonies with neat colony edges and shiny, moist and smooth surfaces, and carry out primary and secondary seed propagation in LB culture medium. The bacterial solution of the secondary seed is transferred to a shake flask at a ratio of 1:100 (i.e., 1% by volume), and cultured at 180r / min in a 37°C shaking shaker. After culturing to the plateau stage, centrifuge it at 8000r / min in a high-speed refrigerated centrifuge for 3 minutes, discard the supernatant to collect the bacteria, and resuspend the bacteria in physiological saline; take 100μl of the bacterial solution for multiple dilution, and calculate the viable bacterial concentration of the resuspended bacterial solution by viable bacterial count. The next day, according to the viable bacterial count results, adjust the bacterial solution to 6.0×10 7 CFU / mL, 6.0×10 8 CFU / mL, 6.0×10 9 CFU / mL, 6.0×10 10 Subsequently, 0.5% formaldehyde solution was added to the total volume, and the bacterial solution was inactivated at 37°C and 100 r / min in a shaker for 48 h to prepare inactivated antigens.

[0046] Fifty 7-day-old ducks were randomly divided into five groups, each with 10 ducks, namely four vaccine-immunized test groups and one non-immunized control group. Each concentration of vaccine was subcutaneously inoculated at the back of the neck at a dose of 0.3 mL / bird, and the control group was not vaccinated. Venous blood was collected on the 14th day after vaccination and 2.5×10 10 The patients were challenged with Escherichia coli JX164 strain at 100 CFU / feather. The incidence and mortality within 7 days after challenge were observed and recorded, and the serum was separated and the level of IgG in the serum was determined using a duck immunoglobulin G (IgG) enzyme-linked immunosorbent assay kit. The results are shown in Table 2.

[0047] Table 2 - Results of strain immunity test

[0048]

[0049] Experimental results: As shown in Table 2, when the final content of the inoculated antigen reached 1.8×10 9 CFU / feather can achieve a high protection rate, and the IgG antibody level in the serum is also the highest. Therefore, from the perspective of economy, efficiency, and practicality, the optimal bacterial content of the vaccine is determined to be 6.0×10 9 CFU / mL.

[0050] Example 3: Preparation of inactivated avian Escherichia coli O145 vaccine

[0051] Avian Escherichia coli O145 inactivated vaccine (containing 20% ​​volume MONTANIDETM GEL P PR adjuvant): Resuscitate and collect the bacteria according to the above method, and adjust the concentration of the bacterial solution to 7.5×10 9 CFU / mL, take 8mL of the adjusted bacterial solution, add 0.5% formaldehyde solution to the total volume, and inactivate the bacterial solution at 37℃ shaker at 100r / min for 48h; dip the inactivated bacterial solution with a sterile cotton swab and apply it to the LB plate for sterility test. Place the sterile immunogen solution in a stirrer at 200rpm and quickly add 2mL of MONTANIDE TM GEL P PR adjuvant (added within 2 seconds), stirred at 200 rpm for 10 min, final antigen content: 6.0×10 9 CFU / mL. Place the prepared vaccine at 4℃ for 12h to observe whether it is evenly distributed and whether there is stratification, and store it at 2-8℃.

[0052] Avian Escherichia coli O145 inactivated vaccine (containing 30% volume aluminum hydroxide gel adjuvant): adjust the bacterial solution concentration to 8.6×10 9 CFU / mL, take 7mL of the adjusted bacterial solution, add 0.5% formaldehyde solution to the total volume, and inactivate the bacterial solution at 37℃ shaker at 100r / min for 48h; dip a sterile cotton swab into the inactivated bacterial solution and apply it to the LB plate for sterility test. Shake the unopened aluminum hydroxide gel adjuvant vigorously to resuspend it, drop 3mL of aluminum hydroxide into the immunogen solution, and add 0.01% thimerosal at a final concentration (mass concentration), pipette up and down several times to mix, ensure the correct absorption of the antigen by the alum adjuvant (i.e., aluminum hydroxide gel adjuvant), incubate for 5-10min, and the final concentration of the antigen is 6×10 9 CFU / mL. Place the prepared vaccine at 4℃ for 12h to observe whether it is evenly distributed and whether there is stratification, and store it at 2-8℃.

[0053] Safety verification of inactivated vaccine: 60 healthy ducks aged 7 days were randomly divided into 6 groups, 10 ducks in each group. Both vaccines were warmed in a 41°C water bath for 30 minutes before injection. The animals were vaccinated at 1 times (0.3 mL / bird), 2 times (0.6 mL / bird), and 3 times (0.9 mL / bird) of the designed immunization dose. The health status of the animals was observed for 7 consecutive days after vaccination to verify the safety of the vaccine. The results are shown in Table 3.

[0054] Table 3 - Results of inactivated vaccine safety trials

[0055]

[0056] Experimental results: After the vaccine was left to stand for 12 hours, the vaccine was evenly distributed without stratification. As shown in Table 3, no obvious adverse reactions occurred in the ducks of each group within 7 days after immunization, and the survival rate reached 100% at 7 days after immunization. This shows that the two inactivated vaccines prepared by the present invention have good safety.

[0057] Example 4: Vaccine Immunoprotective Test

[0058] Experimental method: 100 healthy 7-day-old ducks were randomly divided into 10 groups, with 10 ducks in each group. The immunization site was subcutaneous in the back of the neck. As shown in Table 4, the immunization dose was 0.3 mL per duck, and the immunization antigen content was 1.8×10 9 CFU / feather. Blank control without any treatment, MONTANIDE TM On the 21st day after immunization, the GEL P PR adjuvant control group was challenged with the same serotype strain and the different serotype strain, respectively. The APEC-119 (O78) challenge dose was 1.83×10 10 CFU / feather, the challenge dose of Escherichia coli JX164 (O145) was 2.62×10 10 CFU / feather. The incidence and mortality of animals in each group within 7 days of challenge were observed and recorded to evaluate the immune protection effect of the vaccine. The results are shown in Table 4.

[0059]

[0060]

[0061] Experimental results: As shown in Table 4, the inactivated vaccine of avian Escherichia coli O145 prepared by the present invention can resist infection by strong strains of the same serotype and provide a higher protection rate, but the cross protection rate is low. TM The vaccine prepared with GEL P PR adjuvant had better immune protection than the vaccine prepared with 30% aluminum hydroxide gel adjuvant. The surviving ducks in the control group were autopsied on the 7th day after the challenge, and E. coli lesions such as fibrinous pericarditis and perihepatitis were observed in all ducks.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A poultry Escherichia coli, which is Escherichia coli ( Escherichia coli )JX164, the deposit number is CCTCCNO: M 20231988.

2. Use of the avian Escherichia coli as claimed in claim 1 in the preparation of an inactivated vaccine of avian Escherichia coli O145 serotype.

3. An inactivated vaccine of avian Escherichia coli O145 serotype, characterized in that: The invention comprises the avian Escherichia coli as claimed in claim 1.

4. The avian Escherichia coli O145 serotype inactivated vaccine according to claim 3, characterized in that In the avian Escherichia coli O145 serotype inactivated vaccine, the antigen content of the inactivated Escherichia coli strain is ≥ 6.0×10 9 CFU / mL.

5. The avian Escherichia coli O145 serotype inactivated vaccine according to claim 3, characterized in that Adjuvants are also included.

6. The inactivated vaccine of avian Escherichia coli O145 serotype according to claim 5, characterized in that The adjuvant is aluminum gel adjuvant or MONTANIDE GEL P PR adjuvant.

7. The inactivated vaccine of avian Escherichia coli O145 serotype as claimed in claim 5, characterized in that The volume fraction of the adjuvant in the avian Escherichia coli O145 serotype inactivated vaccine is 20-30%.

8. A method for preparing an inactivated vaccine of avian Escherichia coli O145 serotype as claimed in any one of claims 3 to 7, characterized in that: It includes the following steps: Preparation of inactivated antigen of avian Escherichia coli strain: inoculating the Escherichia coli strain into LB medium for culture, and performing primary and secondary seed propagation respectively; then transferring culture and performing inactivation treatment; Preparation of vaccine: dilute or concentrate the inactivated bacterial solution to make the number of live bacteria before inactivation ≥ 6.0 × 10 9 CFU / mL; take the inactivated bacterial solution and the adjuvant, mix them evenly, stir and emulsify them to obtain the avian Escherichia coli O145 serotype inactivated vaccine.

9. The method for preparing the inactivated vaccine of avian Escherichia coli O145 serotype as claimed in claim 8, characterized in that: The transfer culture conditions are as follows: the bacterial solution of the secondary seeds is transferred to a shaking flask at a ratio of 1: (80-120), and the bacteria are cultured at 170-190 r / min in a shaking incubator at 35-38° C. After culture reaches the plateau phase, the bacteria are centrifuged at 7000-9000 r / min for 2-4 min in a high-speed refrigerated centrifuge, the supernatant is discarded, the bacteria are collected, physiological saline is used to resuspend the bacteria, 90-110 μL of the bacterial solution is taken for multiple dilution, and the viable bacteria concentration of the resuspended bacterial solution is calculated by counting the viable bacteria.

10. The method for preparing the inactivated vaccine of avian Escherichia coli O145 serotype as claimed in claim 9, characterized in that: The inactivation treatment is specifically as follows: adding a 0.3-0.7% formaldehyde solution by volume to the diluted bacterial solution, and inactivating the bacteria at 35-38° C. in a shaking incubator at 80-120 r / min for 40-60 hours.

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