CN115381932B - Trivalent inactivated vaccine for colibacillosis of birds and preparation method thereof - Google Patents
Trivalent inactivated vaccine for colibacillosis of birds and preparation method thereof Download PDFInfo
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Abstract
The invention provides a trivalent inactivated vaccine for fowl colibacillosis and a preparation method thereof, wherein the vaccine comprises inactivated O109, O36 and O78 three serotypes of escherichia coli and an adjuvant, the three types of escherichia coli are respectively subjected to aeration culture, bacterial liquid is harvested and inactivated, and then the three types of escherichia coli are mixed to prepare the inactivated vaccine, the preservation number of the O109 serotype of escherichia coli is CCTCCM20211519, the preservation number of the O36 serotype of escherichia coli is CCTCCM20211520, and the preservation number of the O78 serotype of escherichia coli is CCTCCM20211518. The immune protection rate of the invention against virulent strains of the serous O109, O36 and O78 serous fowl pathogenic escherichia coli reaches more than 90%. The invention has good immune protection effect on O109, O36 and O78 serous fowl 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 colibacillosis of poultry and a preparation method thereof.
Background
The pathogenic colibacillosis of poultry is that some specific serotypes of pathogenic colibacillosis (pathogenic Escherichia coli) infect chickens of different ages and varieties through various ways, cause various symptoms such as acute septicemia, perihepatitis and the like of the chickens of different ages and varieties, especially have very high infection rate and death rate for chickens, often secondary to various viral and respiratory diseases and the like, and are one of main bacterial diseases affecting the development of chicken industry.
The bird pathogenic escherichia coli (Avian pathogenic Escherichia coli, APEC) has various serotypes, and domestic and foreign researches show that most of pathogenic serotypes of the APEC are the most dominant pathogenic serotypes of the O1, O2, O35, O36 and O78, and the pathogenicity of the APEC is different in different regions and different in epidemic serotypes.
Furthermore, APEC is also a major source of transmission of drug resistance genes through its carrying plasmids and genetic material exchange. Production practice for many years shows that the effect of eliminating susceptibility factors or preventing and controlling the colibacillosis of poultry through medicines is very small, and the quality safety of poultry products is increasingly valued along with the continuous generation of drug-resistant strains and the increase of the problems of drug residues. Therefore, prevention and control of APEC by non-antibiotic methods would be a necessary trend in future development, where vaccination is an important approach to effectively prevent and control avian colibacillosis from occurring and becoming prevalent, reduce contamination of avian products, and ensure food safety.
The genetic engineering vaccine is difficult to ensure due to higher production cost and biological safety, is not as widely applied in actual production as inactivated vaccine, and most of the E.coli inactivated vaccines circulated in the market at present are propolis adjuvant vaccines and are protected against O78 type single serotypes, but E.coli vaccines have poor cross protection capability on different serotypes, so multivalent vaccines are prepared against dominant epidemic serotypes to provide protection. The existing product adopts the propolis adjuvant to prepare the seedlings, but the propolis adjuvant is difficult to generate higher protective force although the safety is better, so the adjuvant which has smaller side effect on poultry and higher protective force after the seedlings are prepared should be searched.
Disclosure of Invention
The invention aims to provide O36, O78 and O109 serotype fowl pathogenic escherichia coli, which has strong pathogenicity, can keep strong immunogenicity after inactivation, and is suitable for preparing medicines for treating fowl colibacillosis. The invention also aims to provide a trivalent inactivated vaccine for avian colibacillosis, which can induce higher antibody level and can protect infection of serotype virulent strains such as Escherichia coli O78, O36, O109 and the like which are currently popular.
The invention adopts the following technical scheme:
an avian pathogenic Escherichia coli strain, escherichia coli (Escherichia coli) EC-YP1907-106, was deposited at the chinese collection for typical cultures at 1, 12 months, 2021, at the following deposit: china, university of Wuhan, which has a preservation number of CCTCC M20211519
Can be used for preventing O109 serotype fowl pathogenic escherichia coli infection, and has a protective effect of more than 90% after one immunization.
The invention relates to an application of a pathogenic escherichia coli strain of poultry in preparing vaccines.
The invention provides an inactivated vaccine for avian pathogenic escherichia coli with the preservation number of CCTCC M20211519.
The invention also provides an inactivated vaccine which is prepared by respectively carrying out aeration culture on three kinds of serotype escherichia coli of O109, O36 and O78, then obtaining bacterial liquid, inactivating the bacterial liquid, and then mixing the bacterial liquid and the bacterial liquid.
In the vaccine provided by the invention, O36 serotype escherichia coli has a collection number of CCTCC M20211520, O78 serotype escherichia coli has a collection number of CCTCC M20211518, and the escherichia coli is all collected in China center for type culture collection (China center for type culture collection) at 12 months of 2021, wherein the collection addresses are as follows: chinese, university of martial arts, martial arts.
Can be used for preventing O109, O78 and O36 serological fowl pathogenic Escherichia coli infection, and has a protective effect of more than 80% after one immunization.
In the inactivated vaccine for pathogenic escherichia coli of poultry, 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 of the invention, the adjuvant is an aluminum gel adjuvant or a medical white oil adjuvant.
The invention relates to a method for preparing an inactivated vaccine of pathogenic escherichia coli of poultry, which comprises the following steps:
1) Preparation of inactivated antigen of avirulent E.coli strain
Inoculating the avian pathogenic escherichia coli strain into an LB (LB) culture medium for culture, and respectively carrying out primary seed propagation and secondary seed propagation; then transferring and culturing, and carrying out inactivation treatment;
2) Formulation of vaccines
Diluting or concentrating the inactivated bacterial liquid to make the number of viable bacteria before inactivation greater than or equal to 1.2X10 10 CFU/ml; taking inactivated bacterial liquid and an adjuvant according to the following proportion of 3: mixing evenly in a volume ratio of 1, stirring and emulsifying to obtain the avian pathogenic escherichia coli inactivated vaccine.
Specifically, the condition of transfer culture in the step 1) is that the volume ratio of the secondary seed liquid is 1:100 is transferred into a shaking flask, a shaking table is vibrated for 180r/min at 37 ℃, and after fermentation is carried out for three hours, OD is measured every 0.5 hour 600 When fermentation liquor OD 600 When the amount was 1.3, fermentation was stopped. Centrifuging at 4deg.C for 15min at 8000r/min by a high-speed refrigerated centrifuge, discarding supernatant, re-suspending bacterial sludge by using 1/20 volume of physiological saline of original volume fermentation liquor, fully shaking and uniformly mixing, taking 100 μl bacterial liquid for multiple dilution, and determining final concentration of each bacterial liquid by viable bacteria counting;
the inactivation treatment adopts the bacteria liquid to add formaldehyde solution with the volume ratio of 0.5 percent, and the shaking table is used for 100r/min inactivation for 48 hours at the temperature of 37 ℃.
The invention uses EC-YG1908-580, EC-YP1907-106 and EC-YP1907-54 strains as strains for preparing vaccine, uses medical white oil or aluminum gel as adjuvant, and determines the safety, the immune dose, the vaccine antigen content and the immune protection period of the vaccine by animal experiments of different batches so as to achieve the optimal immune protection effect. And the immune effect of the vaccine is superior to that of similar products through mortality and morbidity of chicken groups in immune protection experiments and through evaluation data of antibody levels in the chicken groups by enzyme-linked immunosorbent assay.
The beneficial effects of the invention are as follows:
1. the vaccine candidate strain is derived from the separated strains of a plurality of disease materials in province and is determined together through serological typing, virulence gene detection analysis and chicken pathogenicity test;
2. the strain of the vaccine is inactivated by formaldehyde solution;
3. the adjuvant of the vaccine can be medical white oil adjuvant or aluminum gel adjuvant, and both can produce good protection effect on chicken flock after the vaccine is prepared, and the growth performance of the chicken flock is not affected.
4. The vaccine has a protective effect on laying hens and broilers.
In conclusion, the monovalent seedlings of the avian escherichia coli O9, O78 and O109 can achieve better protection effect on the infection of homotype strains, but the same single serotype avian escherichia coli is only protected, and the cross protection effect is not ideal. Therefore, aiming at the currently main popular O78, O9 and O109 serotypes, the trivalent inactivated vaccine for avian colibacillosis is developed so as to protect the infection of the three serum colibacillosis at the same time, and the antibody production time and maintenance level after the immunization of a white oil adjuvant or an aluminum gel adjuvant vaccine with lower viscosity are superior to those of a propolis adjuvant.
In conclusion, the trivalent inactivated vaccine of the avian escherichia coli developed by the invention has good immunogenicity, and the result after animal immunization shows that the trivalent inactivated vaccine is safe to chickens, can induce higher antibody level, can protect the currently popular escherichia coli O78, O36, O109 and other serotype virulent strains from being attacked, generates ideal protection effect, and has wide application prospect.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The instruments, reagents, etc. used in the examples are conventional instruments, reagents, etc. existing in the prior art, and are commercially available unless otherwise specified.
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 pathogenic chicken organs and tissues, and after complete inactivation, the pathogenicity is reduced, but the immunogenicity is maintained, and the bacteria are used for preparing inactivated vaccines so as to provide more perfect immune protection for poultry. Colony morphological characteristics of the above strains on the medium of MAIKAI: the fresh peach color or reddish color has deep peach color, round shape, flat shape, regular edge, smooth surface and moist colony center. The nutrient agar culture medium is round, has neat edge, smooth surface, translucency and small bulges. The biochemical characteristics are as follows: the methyl red test was positive and indole production and lactose fermentation were positive.
The present invention will be described in detail with reference to examples.
Example 1: separation and purification of bacterial strain and physicochemical property analysis
(1) Separation and identification of escherichia coli of birds
The method comprises the steps of aseptically collecting organs such as heart, liver and spleen of a eliminated chicken due to colibacillosis of poultry, dipping a tangent plane by an inoculating loop, marking three areas on a MAIKAI agar plate, selecting single colony to carry out enrichment, purifying and culturing, and then determining that the mixture of bacterial liquid and 50% glycerol 1:1 is frozen in a refrigerator at the temperature of minus 20 ℃ after colibacillosis is determined to be the colibacillosis.
(2) Identification of avian E.coli serotypes
Coli which has been purified and preserved with glycerol was purified in a volume ratio of 1:100 is transferred into 1ml of LB culture medium, shake cultured on a shaking table at 37 ℃ and 180r/min for 12 hours to activate and increase bacteria, the bacteria increasing liquid is used as a template for serotyping, and the serotype identification is carried out on 352 strains of escherichia coli by a slide agglutination method, and part of main serotypes are shown in table 1. Among the dominant serotypes are the O78, O109, O36 serotypes, as shown in table 1.
TABLE 1 part serotype identification results
(3) Avian escherichia coli virulence gene detection
The detection of 18 pairs of virulence genes, including adhesin-related genes, was performed on the dominant serum strains screened and the pathogenic serum strains reported in the literature: fimH, tsh; iron-related genes: iucC, iutA, iroN, sitA, feoB, iucD; heat stable toxins: astA, protector related genes: traT, iss, ompT, cvaC; pilus genes: fimC; yersinia virulence island: irp2, fyuA; other virulence genes: colV, hlyE. The detection rate of each virulence gene is counted, and the dominant serotype escherichia coli strains are further screened according to the detection number of the virulence genes.
Of the 18 virulence genes, the fimH and feoB detection rate is 100%; iroN, sitA, iss, fimC, astA the detection rate of the 5 virulence genes is more than 90 percent; iucC, iutA, traT, ompT, iucD the detection rate of the 5 virulence genes is more than 80 percent; the detection rate of 2 virulence genes of colV and hlyE is 0 percent; the remaining virulence genes were lower in detection rate, and the virulence genes with higher detection rate suggested that the virulence genes could be conserved virulence genes, and specific detection results are shown in table 2.
TABLE 2 detection results of virulence genes of E.coli of O35, O36, O78, O109 serotypes
Example 2: screening of vaccine candidate strains
Chick pathogenicity test
As shown in Table 3, 6 strains of D580 (O78), D573 (O78), D224 (O35), D106 (O109), D104 (O36) and D54 (O36) were selected based on the number of serotype and virulence gene detections, and a chick pathogenicity test was performed to select a virulent strain as a vaccine candidate strain.
TABLE 3 pathogenicity test results for O109, O78, O36, O35 serotype chicks
According to the criteria of the pathogenicity of the strains, the 6 strains all belong to highly pathogenic strains, wherein 3 APEC with stronger virulence, namely D580 (O78), D106 (O109) and D54 (O36), are selected as vaccine preparation strains, which are named as Escherichia coli EC-YG1908-580, escherichia coli EC-YP1907-106 and Escherichiacoli EC-YP1907-54 respectively. And is preserved in China center for type culture Collection (China) at 12 th month 1 of 2021 with the following preservation addresses: china, wuhan university, and preservation numbers are CCTCC M20211518, CCTCC M20211519 and CCTCC M20211520 respectively.
Example 3: immune efficacy detection of trivalent inactivated vaccine of escherichia coli O36, O78 and O109
(1) Preparation of monovalent seedlings of Escherichia coli O36, O78 and O109
Inoculating activated EC-YG1908-580, EC-YP1907-106 and EC-YP1907-54 into LB for resuscitation, streaking, inoculating onto LB plate, placing into incubator, shake culturing at 37deg.C overnight, selecting single colony on the next day, respectively breeding primary and secondary seeds, inoculating bacterial liquid of the secondary seeds into shake flask at 1:100, shake shaking table 180r/min at 37deg.C for enrichment fermentation, fermenting for three hours, and measuring one-side OD every 0.5 hr 600 When OD 600 Stopping fermentation when the bacterial strain reaches 1.3, centrifuging at the temperature of 4 ℃ by a high-speed refrigerated centrifuge for 15min at 8000r/min to collect bacteria, discarding supernatant, adopting physiological saline with the volume of 1/20 of the original volume of fermentation liquor to resuspend bacterial mud, taking 100 mu l of bacterial liquid for each serotype bacterial strain after full shaking for dilution, and determining the bacterial amount of each bacterial liquid finally by viable bacteria counting.
Adding 0.5% formaldehyde solution of the total volume into 3 serotype strains with adjusted concentration, placing the bacterial solution into a shaking table at 37 ℃ for 100r/min for inactivation for 48 hours, and carrying out aseptic inspection when the bacterial solution is inactivated for 24 hours and the bacterial solution is inactivated for 48 hours, wherein the aseptic inspection is carried out according to the annex of the current Chinese veterinary pharmacopoeia, so that the aseptic growth is ensured, and the inactivation is complete.
Preparing seedlings by using aluminum gel adjuvant: mixing bacterial liquid after the inactivation of each serotype with an aluminum gel adjuvant according to the volume ratio of 3:1, placing the mixture into a sealed container, placing the container on a shaking table at 4 ℃, fully and uniformly mixing for 12 hours at 300r/min, adding thiomersal with the final concentration of 0.01%, and obtaining each monovalent inactivated vaccine of the avian escherichia coli after the emulsification is finished, wherein the content of each strain of the vaccine is 1.2 multiplied by 10 10 CFU/ml。
Seedling preparation by white oil adjuvant: mixing bacterial liquid after the inactivation of each serotype with white oil adjuvant according to the volume ratio of 3:1, filling the mixture into a sealed container, fully emulsifying the vaccine by a constant-speed powerful electric stirrer, adding thiomersal with the final concentration of 0.01%, and uniformly mixing to obtain the monovalent inactivated vaccine of the avian escherichia coli, wherein the content of each strain of the vaccine is 1.2 multiplied by 10 10 CFU/ml。
(2) Preparation of trivalent inactivated vaccine for colibacillosis of birds
Inoculating activated EC-YG1908-580, EC-YP1907-106 and EC-YP1907-54 into LB for resuscitation, streaking, inoculating onto LB plate, placing into incubator, shake culturing at 37deg.C overnight, selecting single colony on the next day, respectively breeding primary and secondary seeds, inoculating bacterial liquid of the secondary seeds into shake flask at 1:100, shake shaking table 180r/min at 37deg.C for enrichment fermentation, fermenting for three hours, and measuring one-side OD every 0.5 hr 600 When OD 600 Stopping fermentation when the bacterial strain reaches 1.3, centrifuging at the temperature of 4 ℃ by a high-speed refrigerated centrifuge for 15min at 8000r/min to collect bacteria, discarding supernatant, adopting physiological saline with the volume of 1/20 of the original volume of fermentation liquor to resuspend bacterial mud, taking 100 mu l of bacterial liquid for each serotype bacterial strain after full shaking for dilution, and determining the bacterial amount of each bacterial liquid finally by viable bacteria counting.
Adding 0.5% formaldehyde solution of the total volume into 3 serotype strains with adjusted concentration, placing the bacterial solution into a shaking table at 37 ℃ for 100r/min for inactivation for 48 hours, and carrying out aseptic inspection when the bacterial solution is inactivated for 24 hours and the bacterial solution is inactivated for 48 hours, wherein the aseptic inspection is carried out according to the annex of the current Chinese veterinary pharmacopoeia, so that the aseptic growth is ensured, and the inactivation is complete.
Preparing seedlings by using aluminum gel adjuvant: mixing the bacterial solutions subjected to the inactivation of the 3 serotypes together in equal volume, mixing with an aluminum gel adjuvant according to the volume ratio of 3:1, placing the mixture into a sealed container, placing the container on a shaking table, fully and uniformly mixing at the temperature of 4 ℃ for 12 hours at the speed of 300r/min, adding thiomersal with the final concentration of 0.01%, and obtaining the trivalent inactivated vaccine of the aluminum gel adjuvant for the avian colibacillosis after the emulsification is finished, wherein the content of each strain of bacteria in the vaccine is 1.2x10% 10 CFU/ml。
Preparing seedlings by using an oil adjuvant: mixing the bacterial solutions subjected to the inactivation of the 3 serotypes together in equal volume, mixing the bacterial solutions with an oil adjuvant according to the volume ratio of 3:1, fully emulsifying the vaccine by a constant-speed powerful electric stirrer, adding 0.01% of merthiolate into the vaccine, and obtaining the trivalent inactivated vaccine of the avian colibacillosis oil adjuvant after the emulsification is finished, wherein the content of each strain of bacteria in the vaccine is 1.2 multiplied by 10 10 CFU/ml。
(3) Safety test
As shown in table 4, the safety test was performed on each monovalent vaccine of escherichia coli O36, O78, O109 and trivalent inactivated vaccine of escherichia coli O36, O78, O109, and unqualified batches were removed to ensure vaccine safety.
TABLE 4 safety test of inactivated E.coli vaccine for poultry
(4) Immunoprotection test
Vaccine immunization procedure, in which immunization was performed once subcutaneously via the neck at 14 days of age, the vaccine concentration was 1.2X10 per ml of vaccine 10 CFU/ml, immunization dose 0.2ml. After 14 days of immunization, the EC-YG1908-580, EC-YP1907-106 and EC-YP1907-54 were intramuscularly injected via pectoral muscle at 1.0x10 10 CFU/CFU. A total of 3 experiments were performed with 10 chickens per batch and the average results are shown in Table 5. The survival of a few offending chickens is generally considered to be caused by individual differences and is not protective.
TABLE 5 monovalent vaccine immunoprotection rates of E.coli O36, O78, O109 in birds
The monovalent seedlings of the avian escherichia coli O9, O78 and O109 can achieve better protection effect on the infection of homotypic strains, but only protect the same single serotype avian escherichia coli, and do not form effective cross protection on other serotypes.
TABLE 6 immune protection rate of trivalent inactivated vaccine against E.coli in first batch
TABLE 7 immune protection rate of trivalent inactivated vaccine against E.coli in second batch
TABLE 8 immune protection rate of trivalent inactivated vaccine against E.coli in third batch
Three batches of trivalent inactivated E.coli vaccine were used to immunize SPF chickens as shown in tables 5-8, respectively. After the toxicity is removed, the trivalent inactivated vaccine of the two adjuvants has obvious protection effect on chicken flocks, and the effect is better than that of monovalent vaccine.
TABLE 9 immune protection rate of trivalent inactivated vaccine against infection with homotype and heterotype strains
Table 10 compares the immune protective effects with the competitive products
The immune protection test is carried out on two competing products and the vaccine prepared by the invention to compare the protection effect, and the competing products and the vaccine prepared by the invention only have O78 type escherichia coli as the same serotype, so that the O78 type escherichia coli is only adopted for toxicity attack. The results show that: the immune protection effect of the two vaccines prepared by the invention is obviously better than that of two competing products. Chicken colibacillosis propolis inactivated vaccine (O78 serotype), and the product of the cholera escherichia coli bivalent inactivated vaccine is produced by Shandong Hua Hong bioengineering Co.
Table 11 compares the immune protection period of the bid
The two competing products and the two adjuvant E.coli inactivated vaccines of the invention are subjected to detoxification 4 months after immunization, and the results show that the protection effect of the vaccine prepared by the invention is obviously better than that of the competing products.
The trivalent inactivated vaccine of the avian escherichia coli developed by the invention has good immunogenicity, and the result after animal immunization shows that the trivalent inactivated vaccine is safe to chickens, can induce and generate higher antibody level, can protect the currently popular escherichia coli O78, O36, O109 and other serotype virulent strains from being attacked, generates ideal protection effect, and has wide application prospect. Trivalent inactivated vaccine for colibacillosis of birds to achieve the purpose of protecting the infection of the three kinds of serum colibacillosis at the same time.
Claims (6)
1. The trivalent inactivated vaccine for the avian colibacillosis comprises inactivated O109, O36 and O78 three serotypes of avian pathogenic escherichia coli, wherein three types of avian pathogenic escherichia coli are respectively subjected to aeration culture, bacterial liquid is harvested and then inactivated, and the three types of avian pathogenic escherichia coli are mixed to prepare the inactivated vaccine, wherein the preservation number of the O109 serotype of avian pathogenic escherichia coli is CCTCC M20211519, the preservation number of the O36 serotype of avian pathogenic escherichia coli is CCTCC M20211520, and the preservation number of the O78 serotype of avian pathogenic escherichia coli is CCTCC M20211518.
2. The vaccine of claim 1, wherein the antigen content of each inactivated avian pathogenic escherichia coli strain in the trivalent inactivated vaccine is 1.2 x 10 or more 10 CFU/ml。
3. The vaccine of claim 2, further comprising an adjuvant.
4. A vaccine according to claim 3, wherein the adjuvant is an aluminium gel adjuvant or a medical white oil adjuvant.
5. A method for preparing a trivalent inactivated vaccine against avian colibacillosis according to any one of claims 1 to 4, characterized in that it comprises the following steps:
1) Preparation of inactivated antigen of avirulent E.coli strain
Inoculating the avian pathogenic escherichia coli strain into an LB (LB) culture medium for culture, and respectively carrying out primary seed propagation and secondary seed propagation; then transferring and culturing, and carrying out inactivation treatment;
2) Formulation of vaccines
Diluting or concentrating the inactivated bacterial liquid to make the number of viable bacteria before inactivation greater than or equal to 1.2X10 10 CFU/ml; taking inactivated bacterial liquid and an adjuvant according to the following proportion of 3: and (3) uniformly mixing, stirring and emulsifying the mixture according to the volume ratio to obtain the trivalent inactivated vaccine for the avian colibacillosis.
6. The method according to claim 5, wherein the condition of transfer culture in step 1) is that the volume ratio of the secondary seed liquid is 1:100 is transferred into a shaking flask, a shaking table is vibrated for 180r/min at 37 ℃, and after fermentation is carried out for three hours, OD is measured every 0.5 hour 600 When fermentation liquor OD 600 Stopping fermentation when the measurement value is 1.3, centrifuging at 8000r/min at 4deg.C for 15min by a high-speed refrigerated centrifuge, and discarding supernatant to obtain 1% of original volume fermentation liquorRe-suspending the bacterial mud by using physiological saline with the volume of/20, fully vibrating and uniformly mixing, taking 100 mu l of bacterial liquid for multiple dilution, and determining the final concentration of each bacterial liquid by viable bacteria counting; the inactivation treatment adopts the bacteria liquid to add formaldehyde solution with the volume ratio of 0.5 percent, and the shaking table is 100r/min for inactivation for 48 hours at the temperature of 37 ℃.
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