JP2013116869A - Live vaccine against infectious bursal disease containing cell internal virus as main component - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new vaccine against IBD (infectious bursal disease) and a method for immunizing poultry using the vaccine.SOLUTION: There are provided live vaccine against infectious bursal disease (IBD) containing cells infected by IBD and a method for immunizing poultry including injection of the vaccine into growing eggs.

Description

  The present invention relates to a live vaccine against IBD comprising cells infected with infectious bursal disease (IBD) virus, and a method for immunizing poultry using the same.

  Infectious bursal disease (hereinafter referred to as “IBD”) is commonly referred to as Gamboro disease and is a viral disease that develops in poultry such as chickens and turkeys due to IBD virus infection. Is specified.

  The IBD virus belongs to the biraviviridae abivirnavirus, and infection causes necrosis and inflammatory reaction of lymphoid tissue and a bursa of Fabricius (hereinafter referred to as “F sac”). Therefore, when infected with the IBD virus, immune function is reduced, host resistance and various vaccine effects are reduced, and pathogenicity is enhanced and / or other diseases are induced. In the case of IBD virus infection, in addition to the direct damage caused by the onset of IBD, there is also economic damage due to a decrease in feeding performance / productivity, etc. Therefore, it is very important to control the IBD virus in this field. Is being viewed.

  Today, live vaccines have been developed and widely used as a measure against IBD. A live vaccine is produced, for example, by inoculating an IBD virus into a cultured chicken egg or chicken embryo primary cultured cell and culturing, and collecting the IBD virus by filtration or centrifugation (Patent Documents 1 and 2). The live vaccine is administered several times by injection into hatched chicks or by spray vaccination, aerosol vaccination or drinking water vaccination.

  However, vaccines are susceptible to transfer antibodies, and the degree of disappearance of transfer antibodies varies among individuals, and depending on the method of vaccination, all individuals cannot be vaccinated uniformly. In addition, the effect of vaccination varied and became a problem. Furthermore, vaccination had to be carried out several times for each chick, which was a heavy burden on the practitioner. Therefore, in this field, there has been a strong demand for the development of an effective IBD vaccine that is not affected by the transfer antibody and an efficient administration method of the IBD vaccine.

Marek's disease (hereinafter referred to as “MD”) is a viral disease that develops in poultry such as chickens and pupae caused by infection with MD virus. Live vaccines have been developed and are widely used as a measure against MD. MD virus originally has very strong properties associated with cells, and when virus is released to the outside of the cell, the infectivity and proliferation properties are extremely reduced. To that end, live vaccines are manufactured as intracellular dosage forms. It has been reported that MD can be controlled very efficiently and effectively by inoculating a live chicken egg with the live vaccine (Patent Document 3).
On the other hand, to date, no live vaccine of IBD virus having an endogenous dosage form has been produced.

Japanese Patent Laid-Open No. 10-327855 Japanese Patent Laid-Open No. 2002-95485 WO00 / 35476

  An object of the present invention is to provide a novel vaccine against IBD and a poultry immunization method using the vaccine.

  As a result of intensive studies in order to solve the above-mentioned problems, the present inventors have efficiently and effectively used poultry by inoculating a developing egg with a live vaccine against IBD containing cells infected with an IBD virus. It was found that immunization was possible, and the present invention was completed.

That is, the present invention includes the following.
[1] A live vaccine against IBD comprising cells infected with infectious bursal disease (IBD) virus.
[2] The vaccine of [1], wherein the IBD virus is an attenuated strain.
[3] The vaccine of [1] or [2], wherein the cells are chicken embryo fibroblast (CEF) cells.
[4] A method for immunizing poultry, comprising inoculating a developed egg with the vaccine of any one of [1] to [3].
[5] The immunization method according to [4], wherein the poultry are chickens.
[6] The immunization method according to [5], wherein the developing egg is 17 to 19 days old.

  According to the present invention, it is possible to provide a novel vaccine against IBD and an efficient and effective poultry immunization method using the vaccine.

The present invention relates to a live vaccine against IBD comprising cells infected with an IBD virus.
As the “IBD virus” in the present invention, a wide variety of virus strains such as wild strains, conventionally known isolates, and mutants thereof can be used without particular limitation.

  Preferably, the IBD virus is an attenuated strain. The attenuated strain may be isolated from the field, or may be a mutant strain that has been artificially attenuated by mutation treatment or genetic recombination. Traditionally, the extent of attenuation of attenuated strains used in live IBD vaccines depends on the degree of toxicity (lymphopening) and the degree of immunosuppression caused by it, in order of increasing toxicity ( It is classified into three types (Mediate to Inter Mediate), medium poison for chicks (Inter Mediate), and large chicks (Inter Mediate to Hot) (animal biological preparation standards). In the present invention, it is preferable to use a virus strain classified into chicks (Mediate to Inter Mediate). The “virus strain classified as chick (Mediate to Inter Mediate)” means a virus strain that does not cause immunosuppression even when administered to chicks 1 to 4 days of age. As attenuated strains of IBD virus, Lukert-BP (LKT-BP), SAL, D-78, OH, Variabt-A, 2512, 2512G, O3, O3BF, FK-78 , K strains, V877 strains, 228E strains, S706 strains, IQ strains, MB-1B strains, TY2C strains and the like are already known, and these may be used in the present invention (particularly limited to these). Not)

  In the present invention, the “cell” is not particularly limited as long as it is highly sensitive to IBD virus and can culture a large amount of IBD virus. Such cells include primary cell cultures such as duck embryo fibroblasts (DEF), chicken embryo fibroblasts (CEF), chicken embryo hepatocytes (CEL), Vero cell line, BGM-70 cell line, Examples include, but are not limited to, cell lines such as RK-13 cell line, MDBK cell line, QT-35 cell line, QM-7 cell line, and LMH cell line. Preferred are cells derived from birds, and particularly preferred are chicken embryo fibroblasts (CEF).

  By inoculating the cells with the IBD virus and culturing, the “cells infected with the IBD virus” in the present invention can be obtained. The IBD virus grows in “cells infected with IBD virus” and is produced from the cells. As the medium and culture conditions used for culturing the infected cells, those generally used for cell culture can be appropriately selected and adjusted. For example, it is appropriately adjusted depending on factors such as the type and amount of cells used, the amount of inoculated virus, medium composition, etc., but at a culture temperature of 35 to 40 ° C. for about 1 to 4 days, preferably IBD virus Can be cultured until just before the cytopathic effect (CPE) is observed.

  After completion of the culture period, the cells can be collected by a commonly used technique. That is, it can be treated with a restriction enzyme such as trypsin, washed by centrifugation, recovered in an appropriate medium, and used as a live vaccine stock solution. The live vaccine stock solution may be diluted with an appropriate physiological buffer (phosphate buffered saline (PBS), Tris buffered saline (TBS), etc.) as necessary. The live vaccine stock solution can be stored frozen by adding a cryoprotectant. As the cryoprotectant, those generally used for cryopreservation of cells can be used. For example, dimethyl sulfoxide (DMSO), glycerol and the like can be used, although not particularly limited.

The live vaccine against IBD of the present invention is inoculated into a fowl's developing egg.
In the present invention, `` poultry '' means all birds that are infected with IBD virus and are at risk of developing IBD, and examples include, but are not limited to, chickens, ducks, quails, ducks, and turkeys. Not. Preferably, it is a chicken.

  In the present invention, “development egg” means a developing egg from fertilization until the chick hatches. Growth eggs are preferably those in the latter half of the incubation period, and in the case of chickens, they are 15-20 days old, preferably 17-19 days old. At this time, the chicks retain high migrating antibodies, and the live IBD vaccine produced by a conventionally known method (i.e., inoculated into the primary cultured cells of embryonated chicken eggs or chicken embryos, filtered or filtered) Even when inoculated with the IBD live vaccine produced by centrifugation and recovery, the vaccine strain is not grown and the vaccine becomes ineffective.

  Upon inoculation of the developing egg, the live vaccine of the present invention is dissolved in an appropriate solution for lysis (after thawing if stored frozen). Examples of the dissolving solution include, but are not limited to, a conventionally known dissolving solution for Marek's disease (MD) vaccine, tryptone phosphate broth (TPB), and the like.

  Inoculation of the live vaccine to the developing egg can be performed using a technique generally used for vaccination of the developing egg. That is, an inoculation needle having an appropriate size (for example, a 22-28 gauge needle) is inserted from the air chamber side of the developing egg and injected or injected into the amniotic cavity and / or the allantoic cavity. An automatic inoculation device may be used. The live vaccine of the present invention can be inoculated together with other vaccines (for example, MD vaccine etc.), and the live vaccine of the present invention and other vaccines may be inoculated simultaneously. Here, “simultaneous inoculation” means not only when the live vaccine of the present invention and other vaccines are inoculated together, but also after the inoculation of either vaccine in the presence of the immunogen contained in the vaccine. This includes the case of inoculating one vaccine.

The inoculation amount of the live vaccine can be administered at a dose of 10 ² to 10 ⁹ TCID ₅₀ , preferably 10 ³ to 10 ⁶ TCID ₅₀ per egg.

  As described in detail in the Examples below, it is possible to obtain an effective antibody titer against IBD virus from the very early stage after hatching by inoculating a live egg against the IBD of the present invention on the eggs, and at least 21 after hatching. High effective antibody titers against IBD virus can be maintained on the day, preferably about 6-8 weeks after hatching. Usually, chicks immediately after hatching are protected by the transfer antibody, and the transfer antibody gradually decreases and disappears approximately 18 to 24 days after hatching. After the disappearance of the transfer antibody, autoantibodies gradually increase due to natural infection and protect the chicks. By inoculating a developing egg with a live vaccine against IBD of the present invention, a high effective antibody titer against IBD virus can usually be obtained even when the transferred antibody is reduced to disappear, and chicks are more effectively treated than IBD virus. Can defend. In general, chicks of about 10 weeks after hatching have a reduced sensitivity to IBD virus, and even if infected, the effect is not so great. By inoculating a developing egg with the live vaccine against IBD of the present invention, a high effective antibody titer against IBD virus can be maintained even at about 6 to 8 weeks after hatching, until the sensitivity to IBD virus decreases. Can protect chicks more effectively than IBD virus.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the technical scope of this invention is not limited to a following example.

Example 1: Preparation of IBD live vaccine Inoculated with IBD virus Lukert-BP strain on chicken embryo fibroblasts that formed a monolayer, until just before cytopathic effect (CPE) by IBD virus was observed (approximately 2 days), Incubate at 37 ℃. Disperse chicken embryo fibroblasts using 0.5 W / V% trypsin solution, and wash and collect the cells by centrifugal precipitation. The collected cells are resuspended in a stabilizer containing frost damage inhibitor (dimethyl sulfoxide), dispensed and sealed in ampoules, and then gradually frozen to -196 ° C using a program freezer, until -196 ° C until use. Store frozen.

Example 2: Chicken immune material and method using live IBD vaccine)
1． Materials Vaccine: Live IBD vaccine prepared in Example 1 Test egg: 18.5-day-old eggs from SPF chickens (SPAFAS)
Neutralization test virus: Lukert-BP strain neutralizing antibody measurement virus

2. Test method
1) Vaccination method and classification Dissolve the vaccine in MD dissolving solution (Wacchinova Co., Ltd.) to 10 ^4.5 TCID ₅₀ /0.05 mL, then 25 mm (25 G) from the center of the blunt end of 75 test eggs. 0.05 mL / piece was inoculated into the egg (in the amniotic cavity) using a needle (test group). As non-vaccinated control eggs, 30 test eggs were inoculated 0.05 ml of PBS into each egg (amniotic cavity) (control group).

2) Hatching results and clinical observations After confirmation of hatching results and chick health on the day of hatching, 55 birds randomly selected (24 in the control group) were individually identified by wing bands. Clinical symptoms were observed for 21 days with an independent positive pressure isolator.

3) Body weight measurement Body weight measurement was performed on all birds at the time of hatching and on days 1, 3, 5, 7, 14, and 21 after hatching.

4) Necropsy and histopathological examination Five histological examinations were performed in each section, and histopathological examination was performed on F sac / thymus / spleen / liver / bone marrow.

5) Antibody titer measurement Blood samples were collected from 10 birds (consisting of the same individual) on days 7, 14, and 21 after hatching, and the neutralizing antibody titer against IBD virus was measured for the obtained sera. The neutralizing antibody titer was measured by 50% plaque suppression using chicken embryo fibroblasts.

6) Virus recovery test Virus isolation was performed using CEF cells from the target organs and blood derived from 5 birds in the test area on days 1, 3, 5, 7, 14, and 21 after hatching, respectively. That is, each collected target organ and blood was added to phosphate buffered saline to prepare a 30% emulsion, and 0.1 mL thereof was inoculated into CEF cells and cultured at 37 ° C. in a 5% CO ₂ environment for 5 days. Those that showed cytopathic effects specific to IBD virus during the culture period were considered positive for virus isolation. The virus content was measured for the positive specimen for virus separation.

3. Test results
1) Hatching results:
The results are shown in Table 1.
In the control plot, 28 out of 30 (93%) hatched, while in the test plot, 64 out of 75 (85%) hatched. Statistical examination by the χ ² method was performed on the hatching rate of the control group and the test group, but no significant difference was observed.
The normal chick rate was 26/28 (93%) in the control group (one died in the control group and one in the depressed state at the time of hatching), compared to 62 in the test group / 64 (97%) (2 died). The statistical test by the χ ² method was also performed on the normal chick rate, but no significant difference (p <0.05) was observed between the test group and the control group.
This result indicated that vaccination did not affect hatching.

2) Clinical status after hatching:
The results are shown in Table 2.
In the test area, two birds died on the third day after hatching, but no abnormalities were found in the autopsy findings, and the cause of death could not be determined. In addition, 10 birds died 10 days after hatching and died 12 days later. At necropsy, postmortem changes were strong, and the cause of death could not be determined. None of the other chicks showed any abnormal clinical symptoms until the end of the study.
In the control plot, one bird died on day 6 after hatching, but this was due to clogging (no abnormalities were found in autopsy findings).
This result showed that vaccination did not affect the clinical state after hatching.

3) Weight gain:
The results are shown in Table 3.
The weight of the chicks in the test group at the time of hatching was almost equal to the weight of the chicks in the control group, and there was no statistically significant difference (student t test). From day 1 to day 3 after hatching, the weight of the test chicks was significantly lower (p <0.05) than that of the control chicks. There was no significant difference.
This result indicated that vaccination did not affect weight gain after hatching.

4) Pathological anatomy:
The results are shown in Table 4.
In the test chicks, F sac atrophy was observed 5-14 days after hatching, but no F sac atrophy was observed 21 days after hatch. No abnormalities were observed in other organs (liver, spleen, bone marrow) throughout the study period.
On the other hand, no abnormality was observed in the organs of the control chicks throughout the period.

  Table 5 shows the results of detailed analysis of F sac atrophy in chicks in the test section. The F sac was scored for the distribution ratio of follicular lymphocytes, and the transition of lesions was compared. The score criteria are 0: normal (100%) 1: 0-20% lymphocyte disappearance, 2: 20-40% lymphocyte disappearance, 3: 40-60% lymphocyte disappearance, 4: 60-80 % Lymphocyte loss, 5: 80-100% lymphocyte loss.

  On the first day after hatching, slight atrophy changes (average score 1.6) were observed in 4/5 birds. On the third day after hatching, atrophy change was observed in all cases, with an average score of 2.8. In all cases, infiltration of pseudoeosinophils was observed in a small amount. There were no necrotic changes. On day 5 after hatching, all patients had atrophy change, but the atrophy change was weaker than that on day 3 after hatch (mean score decreased to 2.2), and no pseudoeosinophil infiltration or necrotic change was observed. It was. On the 7th day after hatching, the degree of atrophy was less than on the 5th day after hatching, and more follicular lymphocyte regeneration was observed (mean score 1.0). Some have normal follicle formation. After 14 days of hatching, recovery progressed further, with 3/5 birds showing normal follicular structure on day 14 after hatching and 4/5 birds showing normal follicle structure on day 21 after hatching (average score 0.6) . There was no abnormality in the control group throughout the period.

Although the F sac was atrophic after vaccination, it was mildly atrophic and then recovered, indicating that the vaccine has very low pathogenicity.
This result indicates that vaccination does not affect the chick organs / tissues after hatching.

5) Antibody test results are shown in Table 6-1 and Table 6-2.
In the chicks in the test group on day 7 after hatching, the average value of the IBD antibody titer was already 564 times, and all cases had reached 200 times, which is the minimum effective antibody titer (Table 6-1). On day 14 after hatching, the average value increased by 5,236 times, and on day 21 after hatching, the average value further increased by 21,497 times (Table 6-2). On the other hand, according to a conventionally known method, a conventionally known IBD live vaccine (IBD live vaccine (versin 2) Vacchinova Co., Ltd.) is forcibly orally administered to a 0-day-old chick and the antibody titer on the 21st day after hatching is determined. When measured in the same manner as above, the average value of 10 individuals was 9,428 times, which was significantly lower than the antibody titer caused by the vaccination.
This result indicates that the vaccine has a very high immunogenicity.

6) Detection and quantification of virus The results are shown in Tables 7-1 and 7-2.
Virus was detected in all organs examined from day 1 to day 7 after hatching. On the other hand, on day 14 after hatching, the virus was detected only from the spleen, and no virus was detected in all organs on day 21 after hatching (Table 7-1).

When the viral load (infectious titer) was measured for the organ in which the virus was detected, it was shown that a large amount of virus was present in each organ, particularly the F sac, 1 to 3 days after hatching (Table 7-2). ). On the other hand, from 5 to 7 days after hatching, the viral load in each organ decreased.
From this result, it was shown that the virus spread quickly throughout the whole body and proliferated by vaccination in the eggs of the growing chicken.

  From the above, it was shown that inoculation of a vaccine containing an IBD virus-infected cell to a developing egg is safe without affecting the hatched and hatched chickens. In addition, it was shown that by inoculating the developing egg with the vaccine, the virus quickly spreads to the organs throughout the body and can grow. Furthermore, a single inoculation of the vaccine to a developing egg can provide an effective IBD antibody titer early after hatching, and the antibody titer is very high even at least 21 days after hatching. It was shown that chickens can be immunized efficiently and effectively.

  As noted above, to date, no live vaccine of IBD virus having an endogenous dosage form (ie, the form of a virus-infected cell) has been produced. The IBD virus does not have cell-related properties like MD virus, and the conventional live IBD vaccine produced by recovering cultures of embryonated chicken eggs or chicken embryo primary cultured cells From the fact that it had a sufficient effect and the fact that the chicks in the second half of the incubation period have high migrating antibodies, the conventional IBD live vaccine cannot obtain a sufficient effect. A live vaccine comprising cells infected with an IBD virus according to the invention and an immunization method comprising inoculating a developed egg with the vaccine have not been produced and studied. As disclosed herein, according to the present invention, poultry can be immunized against IBD very efficiently and effectively. Therefore, the present invention is expected to contribute in various fields dealing with poultry.

Claims (6)

  A live vaccine against IBD, which includes cells infected with infectious bursal disease (IBD) virus.   The vaccine according to claim 1, wherein the IBD virus is an attenuated strain.   The vaccine according to claim 1 or 2, wherein the cells are chicken embryo fibroblast (CEF) cells.   A method for immunizing poultry, comprising inoculating a developing egg with the vaccine according to any one of claims 1 to 3.   The immunization method according to claim 4, wherein the poultry are chickens.   The immunization method according to claim 5, wherein the developing egg is 17 to 19 days of age.