CN115381932A - Trivalent inactivated vaccine for avian colibacillosis and preparation method thereof - Google Patents
Trivalent inactivated vaccine for avian colibacillosis and preparation method thereof Download PDFInfo
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Abstract
The invention provides an avian colibacillosis trivalent inactivated vaccine and a preparation method thereof, the vaccine contains three serotypes of Escherichia coli and adjuvant of O109, O36 and O78 which are inactivated, the three serotypes of Escherichia coli are respectively subjected to aerated culture and then are inactivated and mixed to prepare the inactivated vaccine, the O109 serotype of Escherichia coli has the preservation number of CCTCCM20211519, the O36 serotype of Escherichia coli has the preservation number of CCTCCM20211520, the O78 serotype of Escherichia coli has the preservation number of CCTCCM20211518. The immunoprotection rate of the invention to the virus attack of serotype O109, O36 and O78 avian pathogenic escherichia coli virulent strains reaches more than 90%. The invention has good immune protection effect on O109, O36 and O78 serotype avian pathogenic escherichia coli strain infection.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a trivalent inactivated vaccine for avian colibacillosis and a preparation method thereof.
Background
Avian pathogenic colibacillosis is one of the main bacterial diseases affecting the development of chicken industry, and is characterized in that certain specific serotype pathogenic Escherichia coli (pathogenic Escherichia coli) can infect chickens of different ages and varieties through multiple ways to cause multiple symptoms of acute septicemia, perihepatitis and the like of chickens of different ages and varieties, especially has high infection rate and death rate to chicks, and is often followed by multiple viral and respiratory diseases and the like.
Avian Pathogenic Escherichia Coli (APEC) has a plurality of serotypes, and domestic and foreign researches show that most of pathogenic sera of the APEC have O1, O2, O35, O36 and O78 types of serogroups as the most main pathogenic serotypes, and the pathogenicity of the APEC is different due to different serotypes popular in different regions.
In addition, APEC is also a major source of transmission of drug resistance genes through its carrier plasmid and genetic material exchange. Production practices for many years show that the effect of preventing and controlling the avian colibacillosis is very slight by eliminating susceptible factors or medicines, and along with the continuous generation of drug-resistant strains and the increase of the problem of drug residues, the quality safety of the avian products is more and more emphasized by people. Therefore, the use of non-antibiotic methods for prevention and control of APEC will be a necessary trend for future development, where vaccination is an important approach to effectively prevent and control the occurrence and prevalence of colibacillosis in poultry, reduce poultry product contamination, and ensure food safety.
Because the production cost of the genetic engineering vaccines is higher, the biological safety is difficult to guarantee, and the genetic engineering vaccines are not as wide as inactivated vaccines in actual production, most of escherichia coli inactivated vaccines which are circulated in the market at present are propolis adjuvant vaccines, and most of the escherichia coli inactivated vaccines are protected against O78 type single serotypes, but the cross protection capability of the escherichia coli vaccines against different serotypes is poor, and therefore, the multivalent vaccines are prepared against dominant epidemic serotypes to provide protection. The existing product adopts the propolis adjuvant to prepare the vaccine, although the safety is better, the propolis adjuvant prepared vaccine is difficult to generate higher protection force, so the adjuvant which generates less side effect on poultry and has higher protection force after vaccine preparation is needed to be searched.
Disclosure of Invention
The invention aims to provide O36, O78 and O109 serotype avian pathogenic escherichia coli which has stronger pathogenicity, can keep stronger immunogenicity after inactivation and is suitable for preparing a medicament for treating avian colibacillosis. The invention also aims to provide the trivalent inactivated vaccine for the avian colibacillosis, which can induce and generate higher antibody level and can protect the infection of serotype virulent strains such as currently epidemic Escherichia coli O78, O36 and O109.
The invention adopts the following technical scheme:
an avian pathogenic Escherichia coli strain, which is Escherichia coli (Escherichia coli) EC-YP1907-106, has been deposited in China Center for Type Culture Collection (CCTCC) at 12 months 1 in 2021, and has the deposition addresses: china, wuhan and Wuhan university with a preservation number of CCTCC M20211519
Can be used for preventing O109 serotype avian pathogenic escherichia coli infection, and after one-time immunization, the protection effect is more than 90%.
The invention relates to an application of avian pathogenic escherichia coli strain in preparation of vaccine.
The invention provides the avian pathogenic escherichia coli inactivated vaccine with the preservation number of CCTCC M20211519.
The invention also provides the vaccine which contains three serotypes of Escherichia coli, namely O109, O36 and O78, wherein the three Escherichia coli are respectively subjected to aeration culture, obtained bacterial liquid is inactivated, and then mixed to prepare the inactivated vaccine.
In the vaccine provided by the invention, the O36 serotype Escherichia coli with the preservation number of CCTCC M20211520 and the O78 serotype Escherichia coli with the preservation number of CCTCC M20211518 are preserved in China Center for Type Culture Collection (CCTCC) at 12 months and 1 days 2021, and the preservation addresses are as follows: china, wuhan university.
Can be used for preventing O109, O78 and O36 serotype avian pathogenic escherichia coli infection, and after one-time immunization, the protection effect is more than 80%.
In the avian pathogenic escherichia coli inactivated vaccine, the antigen content of each inactivated escherichia coli strain is more than or equal to 1.2 multiplied by 10 10 CFU/ml。
The vaccine of the invention also comprises an adjuvant.
In the vaccine, the adjuvant is an alumina gel adjuvant or a medical white oil adjuvant.
The method for preparing the avian pathogenic escherichia coli inactivated vaccine comprises the following steps:
1) Preparation of inactivated antigen of avian pathogenic Escherichia coli strain
Inoculating the avian pathogenic escherichia coli strain into an LB culture medium for culture, and respectively carrying out primary and secondary seed propagation; then, transferring culture and inactivating;
2) Formulated vaccines
Diluting or concentrating the inactivated bacteria solution to make the number of viable bacteria before inactivation greater than or equal to 1.2 × 10 10 CFU/ml; taking the inactivated bacterial liquid and an adjuvant according to the proportion of 3:1 volume ratio, stirring and emulsifying to obtain the avian pathogenic escherichia coli inactivated vaccine.
Specifically, the conditions of the transfer culture in the step 1) are that the volume ratio of the secondary seed liquid is 1: transferring 100 into shake flask, shaking table at 37 deg.C for 180r/min, fermenting for three hours, and measuring OD once every 0.5 hr 600 When fermentation broth OD is reached 600 When the measurement is 1.3, the fermentation is stopped. Centrifuging for 15min at 8000r/min at 4 ℃ by using a high-speed refrigerated centrifuge, discarding supernatant, resuspending bacterial sludge by using 1/20 volume of physiological saline of original volume fermentation liquor, fully shaking and uniformly mixing, diluting 100 mu l of bacterial liquid by multiple ratio, and determining the final concentration of each bacterial liquid by viable count;
and the inactivation treatment is carried out by adding the bacterial liquid into 0.5 percent formaldehyde solution by volume ratio and inactivating for 48 hours at 37 ℃ by a shaking table at 100 r/min.
The invention takes EC-YG1908-580, EC-YP1907-106 and EC-YP1907-54 strains as strains for preparing the vaccine and medical white oil or alumina gel as an adjuvant, and determines the safety, the immunization dose, the content of vaccine antigens and the immunization protection period of the vaccine through animal experiments of different batches so as to achieve the optimal immunization protection effect. And the immunity effect of the vaccine is determined to be superior to that of the similar products through the mortality and morbidity of chicken in an immune protection experiment and the evaluation data of the antibody level in the chicken by an enzyme-linked immunosorbent assay.
The beneficial effects of the invention are as follows:
1. the vaccine candidate strain is derived from a plurality of provincial and municipal disease isolates and is determined by serological typing, virulence gene detection analysis and chicken pathogenicity test;
2. the strains of the vaccine are inactivated by formaldehyde solution;
3. the adjuvant of the vaccine can adopt a medical white oil adjuvant or an alumina gel adjuvant, and both can generate good protection effect on chicken flocks after being prepared into the vaccine, and the growth performance of the chicken flocks is not influenced.
4. The vaccine has protective effect on both laying hens and broilers.
In conclusion, the avian escherichia coli O9, O78 and O109 monovalent seedlings can achieve a good protection effect on infection of homotypic strains, but only protect the avian escherichia coli of the same single serotype, and the cross protection effect is not ideal. Therefore, aiming at the currently mainly prevalent O78, O9 and O109 serotypes, the invention develops the avian colibacillosis trivalent inactivated vaccine to protect the Escherichia coli infection of the three seroses simultaneously, and the antibody generation time and the maintenance level after the vaccine is immunized by selecting the white oil adjuvant or the alumina gel adjuvant with lower viscosity are superior to those of the propolis adjuvant.
In conclusion, the avian escherichia coli trivalent inactivated vaccine developed by the invention has good immunogenicity, and the result after animal immunization shows that the vaccine is safe for chickens, can induce and generate higher antibody level, can protect the attack of serotype virulent strains such as escherichia coli O78, O36 and O109 which are popular at present, generates ideal protection effect, and has wide application prospect.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The instruments, reagents and the like used in the examples are, unless otherwise specified, conventional instruments and reagents known in the art and commercially available.
The avian pathogenic Escherichia coli (Escherichia coli) EC-YG1908-580, EC-YP1907-106 and EC-YP1907-54 provided by the invention are virulent strains obtained by screening and culturing organ tissues of diseased chickens, the pathogenicity is reduced after complete inactivation, but the immunogenicity is kept, and the inactivated vaccine is prepared by using the strains so as to provide more complete immune protection for poultry. The bacterial colony morphology characteristics of the strain on the MacconKa culture medium are as follows: the fresh peach is red or reddish, the center of the colony is dark peach red, round, flat, neat in edge, smooth and moist in surface. The culture medium is round, neat in edge, smooth in surface, semitransparent and small in bulge. The biochemical characteristics are shown as follows: methyl red test is positive, indole production and lactose fermentation are positive.
The present invention will be described in detail with reference to examples.
Example 1: separation and purification of strain and analysis of physicochemical properties
(1) Separation and identification of avian Escherichia coli
Aseptically collecting organs such as hearts, livers, spleens and the like of eliminated chickens caused by avian colibacillosis, dipping an inoculating loop into a section, scribing three zones on a Macconkey agar plate, selecting a single colony, enriching, purifying and culturing, and then performing sequencing identification to determine that the Escherichia coli is mixed with 50% of glycerol 1, and then freezing the mixture in a refrigerator at the temperature of-20 ℃ for short-term storage.
(2) Identification of avian Escherichia coli serotype
Coli which had been purified and preserved with glycerol was cultured in a volume ratio of 1:100 are transferred into 1ml LB culture medium to be shake-cultured for 12h on a shaking table at 37 ℃ and 180r/min for activation and enrichment, enrichment liquid is used as a template for serotype classification, 352 strains of escherichia coli are subjected to serotype classification and identification by a slide agglutination method, and part of main serotypes are shown in Table 1. Among the dominant serotypes are O78, O109, and O36 serotypes, as shown in Table 1.
Table 1 partial serotype identification results
(3) Detection of virulence gene of avian escherichia coli
And (3) detecting virulence genes of the selected dominant serum strain and the pathogenic serum strain reported in the literature, wherein the virulence genes comprise adhesin related genes: fimH, tsh; iron-related genes: iucC, iutA, iroN, sitA, feoB, iucD; heat stable toxin: astA, a protectin-related gene: traT, iss, ompT, cvaC; a pilus gene: fimC; yersinia virulence island: irp2, fyuA; other virulence genes: colV, hlyE. And (4) counting the detection rate of each virulence gene, and further screening the dominant serotype escherichia coli strain according to the detection number of the virulence genes.
Among 18 virulence genes, the detection rate of fimH and feoB is 100 percent; the detection rate of 5 virulence genes of iroN, sitA, iss, fimC and astA is more than 90%; the detection rate of 5 virulence genes of iucC, iutA, traT, ompT and iucD is more than 80%; the detection rate of 2 virulence genes of colV and hlyE is 0 percent; the detection rate of other virulence genes is low, and the virulence genes with high detection rate are suggested to be conserved virulence genes, and specific detection results are shown in table 2.
TABLE 2 O35, O36, O78, O109 serotype E.coli virulence gene detection results
Example 2: screening of vaccine candidate strains
Chick pathogenicity test
As shown in Table 3, 6 strains D580 (O78), D573 (O78), D224 (O35), D106 (O109), D104 (O36) and D54 (O36) were selected according to the serotype and the number of detected virulence genes, and subjected to a chicken pathogenicity test, and virulent strains were selected as vaccine candidate strains.
TABLE 3 chick pathogenicity test results for serotype strains O109, O78, O36, O35
According to the judgment standard of the pathogenicity of the strains, the 6 strains are all classified into highly pathogenic strains, wherein 3 APECs with stronger virulence are selected as vaccine preparation strains, namely D580 (O78), D106 (O109) and D54 (O36), and are respectively named as Escherichia coli EC-YG1908-580, escherichia coli EC-YP1907-106 and Escherichia coli EC-YP1907-54. And is preserved in China Center for Type Culture Collection (CCTCC) at 12 months and 1 days in 2021, wherein the preservation addresses are as follows: china, wuhan university, with the preservation numbers of CCTCC M20211518, CCTCC M20211519, and CCTCC M20211520, respectively.
Example 3: detection of avian Escherichia coli O36, O78 and O109 trivalent inactivated vaccine immunity efficacy
(1) Preparation of avian Escherichia coli O36, O78 and O109 monovalent seedlings
Inoculating activated EC-YG1908-580, EC-YP1907-106 and EC-YP1907-54 into LB for resuscitation, streaking and inoculating on an LB plate, placing in an incubator at 37 ℃ for shake culture overnight, selecting single colony for first-stage and second-stage seed propagation in the next day, inoculating the bacterial liquid of the second-stage seed into a shake flask according to a ratio of 1 600 When OD is reached 600 Stopping fermentation when the concentration reaches 1.3, centrifuging for 15min at 8000r/min by a high-speed refrigerated centrifuge at 4 ℃, discarding supernatant, re-suspending bacterial sludge by using normal saline with the volume of 1/20 of the original fermentation liquor, diluting each serotype strain by 100 mu l bacterial liquid in a multiple ratio after fully shaking, and determining the final bacterial quantity of each bacterial liquid by viable count.
Adding 3 serotype strains with adjusted concentration into 0.5% formaldehyde solution, inactivating the bacteria solution in a shaker at 37 ℃ for 48h at 100r/min, performing sterile test when inactivating for 24h and 48h, and performing sterile test according to the appendix of the current Chinese veterinary pharmacopoeia to ensure that no viable bacteria grow and the inactivation is complete.
Preparing seedlings by using an alumina gel adjuvant: mixing the bacterial liquid subjected to serotype inactivation and the alumina gel adjuvant according to a volume ratio of 3 10 CFU/ml。
Preparing a seedling by using a white oil adjuvant: mixing the bacterial liquid subjected to serotype inactivation and a white oil adjuvant according to a volume ratio of 3 10 CFU/ml。
(2) Preparation of trivalent inactivated vaccine for avian colibacillosis
Inoculating activated EC-YG1908-580, EC-YP1907-106 and EC-YP1907-54 into LB for resuscitationThen, streak inoculation is carried out on an LB plate, the plate is placed in an incubator at 37 ℃ for shake cultivation overnight, single colonies are selected the next day for carrying out primary and secondary seed propagation respectively, the bacterium liquid of the secondary seeds is inoculated in a shake flask according to the ratio of 1 600 When OD is reached 600 Stopping fermentation when the bacterial quantity reaches 1.3, centrifuging for 15min at 8000r/min by a high-speed refrigerated centrifuge at 4 ℃, discarding supernatant, re-suspending bacterial sludge by adopting normal saline with the volume of 1/20 of the original volume of fermentation liquor, diluting each serotype bacterial strain in 100 mu l bacterial liquid by times after full oscillation, and finally determining the bacterial quantity of each bacterial liquid by viable count.
Adding 3 serotype strains with adjusted concentration into 0.5% formaldehyde solution, inactivating the bacteria solution in a shaker at 37 ℃ for 48h at 100r/min, performing sterile test when inactivating for 24h and 48h, and performing sterile test according to the appendix of the current Chinese veterinary pharmacopoeia to ensure that no viable bacteria grow and the inactivation is complete.
Preparing seedlings by using an alumina gel adjuvant: the three-valent inactivated vaccine for avian colibacillosis alumina gel adjuvant is prepared by mixing the bacterial liquid in which the 3 serotypes are inactivated in equal volume together, mixing the bacterial liquid with the alumina gel adjuvant according to the volume ratio of 3 10 CFU/ml。
Preparing a seedling by using an oil adjuvant: the method comprises the steps of mixing 3 serotype inactivated bacteria liquids in equal volumes together, mixing the liquids with an oil adjuvant according to the volume ratio of 3 10 CFU/ml。
(3) Safety test
As shown in Table 4, safety inspection is carried out on the avian Escherichia coli O36, O78 and O109 monovalent vaccines and the avian Escherichia coli O36, O78 and O109 trivalent inactivated vaccines, unqualified batches are removed, and the safety of the vaccines is ensured.
TABLE 4 avian Escherichia coli inactivated vaccine safety test
(4) Immune protection assay
Vaccine immunization procedure, one immunization at 14 days of age via cervical subcutaneous route at a vaccine concentration of 1.2X 10 bacteria per ml vaccine 10 CFU/ml, immunization dose 0.2ml. After 14 days of immunization, the toxin is attacked by intramuscular injection of EC-YG1908-580, EC-YP1907-106 and EC-YP1907-54 to the pectoral muscle, and the dose of the toxin attacking is 1.0 multiplied by 10 10 CFU/only. 10 chickens were added to each batch, and 3 experiments were carried out, with the average results shown in Table 5. The survival of a few offending chickens is generally considered to be caused by individual differences and has no protective power.
TABLE 5 avian Escherichia coli O36, O78, O109 monovalent vaccine immunoprotection rates
The avian Escherichia coli O9, O78 and O109 monovalent seedlings can achieve a good protection effect on infection of homotypic strains, but only protect against the same single serotype avian Escherichia coli, and do not form effective cross protection on other serotypes.
TABLE 6 immunoprotection rates of trivalent inactivated vaccine of avian Escherichia coli of first batch
TABLE 7 immunoprotection Rate of the second batch of avian Escherichia coli trivalent inactivated vaccine
TABLE 8 immunoprotection rates of third batch of avian Escherichia coli trivalent inactivated vaccine
Three batches of trivalent inactivated Escherichia coli vaccine were used to immunize SPF chickens, as shown in tables 5-8, respectively. After the virus attack, the trivalent inactivated vaccine of the two adjuvants has obvious protective effect on chicken flocks, and the effect is superior to that of a monovalent vaccine.
TABLE 9 immunoprotection rate of avian Escherichia coli trivalent inactivated vaccine against homotypic and heterotypic bacterial strain infection
TABLE 10 comparison of the immunoprotection Effect of the Competition products
Two competitive products and the vaccine prepared by the invention are selected to carry out an immune protection test to compare the protection effect, and only O78 type escherichia coli is of the same serotype as the competitive products and the vaccine, so that only O78 type escherichia coli is adopted for virus challenge. The results show that: the immune protection effect of the two vaccines prepared by the invention is obviously superior to that of two competitive products. Propolis inactivated vaccine (O78 serotype) for chicken colibacillosis and the product of the combined inactivated vaccine for cholera colibacillosis are produced by Shandong Huahong bioengineering Co., ltd.
TABLE 11 comparison of the immunoprotection periods of the races
The two competitive products and the Escherichia coli inactivated vaccine of the two adjuvants are subjected to challenge 4 months after immunization, and the result shows that the protective effect of the vaccine prepared by the invention is obviously superior to that of the competitive products.
The avian escherichia coli trivalent inactivated vaccine developed by the invention has good immunogenicity, and results after animal immunization show that the vaccine is safe for chickens, can induce and generate higher antibody level, can protect the attack of serotype virulent strains such as escherichia coli O78, O36 and O109 which are popular at present, generates ideal protection effect, and has wide application prospect. The trivalent inactivated vaccine for avian colibacillosis can protect the infection of colibacillosis of the three sera at the same time.
Claims (6)
1. The trivalent inactivated vaccine for colibacillosis of fowl also contains three serotypes of Escherichia coli, O109, O36 and O78, and the inactivated vaccine is prepared through aeration culture of the three serotypes of Escherichia coli, inactivation of the obtained bacteria liquid and mixing, and has the preservation numbers of CCTCC M20211519 and O36 for Escherichia coli and CCTCC M20211520 and CCTCC M20211518 for Escherichia coli of O78.
2. The vaccine according to claim 1, wherein the inactivated vaccine against avian pathogenic Escherichia coli has an antigen content of 1.2X 10 or more in each inactivated Escherichia coli strain 10 CFU/ml。
3. The vaccine of claim 2, further comprising an adjuvant.
4. The vaccine according to claim 3, wherein the adjuvant is an alumina gel adjuvant or a medicinal white oil adjuvant.
5. The method for preparing the trivalent inactivated vaccine against avian pathogenic escherichia coli according to any one of claims 1 to 4, comprising the steps of:
1) Preparation of inactivated antigen of avian pathogenic Escherichia coli strain
Inoculating the avian pathogenic escherichia coli strain into an LB culture medium for culture, and respectively carrying out primary and secondary seed propagation; then, switching culture is carried out, and inactivation treatment is carried out;
2) Formulating vaccines
Diluting or concentrating the inactivated bacteria liquid to make the viable count before inactivation more than or equal to 1.2 × 10 10 CFU/ml; taking the inactivated bacterial liquid and an adjuvant according to the ratio of 3: and (3) uniformly mixing according to the volume ratio of 1, stirring and emulsifying to obtain the avian pathogenic escherichia coli inactivated vaccine.
6. The method according to claim 9, wherein the condition of the transfer culture in step 1) is that the secondary seed solution is mixed in a volume ratio of 1: transferring 100 into shake flask, shaking table at 37 deg.C for 180r/min, fermenting for three hours, and measuring OD once every 0.5 hr 600 When fermentation broth OD 600 When the measurement is 1.3, the fermentation is stopped. Centrifuging for 15min at 8000r/min at 4 ℃ by using a high-speed refrigerated centrifuge, discarding supernatant, resuspending bacterial sludge by using 1/20 volume of physiological saline of original volume fermentation liquor, fully shaking and uniformly mixing, diluting by taking 100 mu l bacterial liquid in a multiple ratio, and determining final concentration of each bacterial liquid by viable count;
and the inactivation treatment is carried out by adding the bacterial liquid into a formaldehyde solution with the volume ratio of 0.5%, and inactivating for 48 hours at 37 ℃ by a shaking table at 100 r/min.
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| CN104774796A (en) * | 2015-04-29 | 2015-07-15 | 中国农业科学院上海兽医研究所 | Inactivated vaccine for poultry pathogenic escherichia coli and preparation method thereof |
| KR20200138570A (en) * | 2019-05-31 | 2020-12-10 | 강원대학교산학협력단 | Endotoxin-reduced Escherichia coli trivalent inactivated vaccine composition for preventing Avian colibacillosis |
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| CN104774796A (en) * | 2015-04-29 | 2015-07-15 | 中国农业科学院上海兽医研究所 | Inactivated vaccine for poultry pathogenic escherichia coli and preparation method thereof |
| KR20200138570A (en) * | 2019-05-31 | 2020-12-10 | 강원대학교산학협력단 | Endotoxin-reduced Escherichia coli trivalent inactivated vaccine composition for preventing Avian colibacillosis |
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| 高玉斌等: "不同地区动物源致病性大肠杆菌的 分子特性与表型分析", 中国动物检疫, vol. 35, no. 9, pages 62 - 66 * |
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