JPH10327855A - Preparation of infectious bursal disease virus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prepare infectious bursal disease(IBD) virus intended to stably obtain at a low cost an IBD vaccine capable of inducing a neutralizing antibody against avian IBD, by infecting avian-derived established cells with IBD virus to effect its proliferation. SOLUTION: This IBD virus is prepared by infecting avian-derived established cells such as LMH cells with IBD virus to effect its Proliferation. It is preferable that an IBD vaccine containing the above IBD virus (antigen component) is administered to poultry to restrain IBD onset; furthermore, it is preferable to prepare such IBD vaccines and mixed vaccines each containing an antigen derived from at least one kind of microbe selected from other virus, mycoplasma, bacteria and parasites.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for preparing infectious bursal disease (IBD) virus. More specifically, the IBD virus is infected to a cell line derived from a bird to propagate the virus, and the IB
A method for preparing D virus.

[0002]

BACKGROUND OF THE INVENTION Infectious bursal disease (IBD) is an acute infectious disease caused by the IBD virus, and is a viral disease characterized mainly by necrosis of lymphatic tissues including the bursa of Fabricius. When infected with the IBD virus, the immune function is reduced, causing the reduction of the immune effect of various vaccines and the induction or enhancement of other diseases due to the reduction of host resistance. IB
Vaccines have been marketed to prevent D. Until now, methods for growing IBD virus to produce IBD vaccines include, for example, a method of inoculating chickens with the IBD virus and growing it in a sac of Fabricius, Inoculation of embryonated chicken eggs and propagation in chorioallantoic membrane and endosperm or allantoic cavity.
F) or a method of proliferating in mammalian cell lines (Diseases of Poultry EIGHTH EDITION 5)
66-576, 1984). In such a conventional method, when chickens are used, a large number of chickens are bred and aseptic sampling of the fabric sac from the chickens. CEF for aseptic collection of chorioallantoic membrane, chicken embryo or allantoic fluid, and, if CEF is used, chicken embryo collection from embryonated hen eggs for use in tissue culture, shredding and enzyme treatment Each operation requires a lot of labor and time. In addition, starting materials of cultured cells or tissues used for virus propagation are different every time a vaccine is manufactured, and it is not easy to maintain a certain quality.

On the other hand, as materials for vaccine production,
If the cell line can be used, it is possible to eliminate the trouble of preparing materials for producing the virus as described above, improve the productivity, and maintain a vaccine of a constant quality. Become. To date, many cell lines derived from chicken tissues have been reported (Witter RL and Commit
tee Avian Pathol. 8, 487-498, 1979). Such cells include cell lines derived from chicken lymphoblasts (lymphoblastoid c).
ell lines) (Akiyama and Kato, Biken J. 17: 105-116,
1974), chicken fibroblastic cells treated with Rous sarcoma virus or carcinogen (Kaaden et al.
In Vitro 18: 827-834,1982; Ogura et al. Gann. 75: 410.
-414,1984, Dinowitz, MJ Natl. Cancer Inst. 58: 307
-312,1977, Lerman et al. J. Natl. Cancer Inst. 57: 2
95-301,1976) and the only epithelial hepatocellular carcinoma-derived LMH cells (hepatocellular cells) treated with carcinogens
(Kawaguchi et al. Cancer Res. 47: 4460-4464,1987)
Etc. However, these cell lines produce the virus used to induce the cell line, and the infectivity of the cell line with the IBD virus is unknown.
There are no reports of chicken cell lines suitable for producing vaccines. Method for Propagating IBD Virus Using Mammalian-Derived Ave Cells (PDLukert Am J Vet Res, Vol 36,
No. 4 1975: 539-540, Published Patent Publication (A) Hei 2-78
634) is disclosed, however, when the cells derived from mammals are used, a virus antigen having a sugar chain different from that obtained when cells derived from birds is used is produced. May have a different antigenicity from the viral antigens. As a result, it is expected to affect the ability to induce immunity as a vaccine.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for preparing an IBD virus using LMH cells, a chicken cell line.

Another object of the present invention is to provide an IBD vaccine containing an IBD virus or an antigen component thereof as a main component, which is obtained by the above method.

Still another object of the present invention is to provide the above-mentioned I
BD vaccine, as well as another virus, mycoplasma,
It is to provide a combination vaccine containing an antigen derived from at least one kind of microorganism selected from bacteria and parasites.

[0007]

Means for Solving the Problems The present inventors have made intensive studies to achieve the above-mentioned object, and as a result, have found that the only epithelial liver of a chicken that is free from contamination of exogenous microorganisms and has good cell proliferation. The present invention has been found that LMH cells, which are cell lines derived from cell carcinoma, are highly sensitive to IBD virus, and that the IBD vaccine prepared using the LMH cells has sufficient immunogenicity against IBD virus. Was completed. That is, conventionally, there was no suitable avian-derived cell line for the production of IBD vaccine, but when the IBD virus was cultured in LMH cells, which are the only cell line derived from chicken epithelial hepatocellular carcinoma, , Chicken fibroblasts (CE
F) that the virus multiplied to the same extent as F), and that chickens vaccinated with the vaccine prepared using this virus solution showed a high neutralizing antibody titer against the IBD virus. It was completed. This finding makes it possible to prepare an IBD vaccine using avian cell lines.

[0008]

The method of the present invention is characterized by an avian cell line used to propagate the IBD virus. As an IBD virus strain for production, for example, SAL strain, D-78 strain, OH strain, Variabt-A strain, 2512 strain, O3
Strains, O3BF strains, FK-78 strains, K strains and the like can be used. Host cells used for virus propagation are those IBD
Any cell in which the virus multiplies and which is an avian-derived cell line that does not produce the impure virus can be used. Preferably, a chicken cell line is used. More preferably, LMH cells derived from only chicken epithelial hepatocellular carcinoma are used.

[0009] The medium used for cell culture may be a medium generally used for tissue culture, for example, Eagle MEM.
A medium is used, to which a medium such as bovine, chicken or goat serum is added in an amount of 0 to 30%, preferably 2 to 5%.

When the virus is propagated, the number of LMH cells is 1 to 2 million, preferably 10
It is prepared so as to have a density of ~ 500,000, and the virus solution is mixed therewith at the same time, and the mixed solution is allowed to stand in a roux bottle or a roller bottle for cultivation. A method such as a floating culture in which cells are attached to a carrier such as a carrier and the cells are suspended and cultured, or a floating culture in which cells are suspended and cultured without using a carrier is used. In addition, only cells were cultured for 1 to
After culturing for 3 days, a virus solution can be inoculated into this and cultured. Cultivation is performed at 30 to 42 ° C, preferably 32
It is performed at a temperature of 〜38 ° C. for 2 to 8 days. When performing large-scale culture using a fermenter, the ion concentration (pH)
From 6 to 8, preferably 6.5 to 7.5, dissolved oxygen (DO)
Under the condition of 1 to 6 ppm, preferably 2 to 4 ppm.

[0011] The live IBD vaccine of the present invention is obtained by centrifuging the culture obtained by the above-mentioned culture method at 1500 rpm, using the supernatant as a virus suspension, adding a stabilizer such as lactose, and freeze-drying the suspension. It is prepared by The IBD-inactivated vaccine is added to the virus suspension,
After inactivating the virus with an inactivating agent such as formalin, glutaraldehyde or beta-propiolactone, an immunostimulant such as aluminum hydroxide, aluminum phosphate, potassium phosphate, mineral oil or non-mineral oil is added. Prepared. The vaccine of the present invention thus prepared is administered to poultry, for example, by a method of intramuscular, subcutaneous or in ovo injection, or by a method of instillation and instillation in the form of a spray or a method of mixing with drinking water to drink. Is done.

The IBD vaccine of the present invention can be used as a combination vaccine. In the case of a live vaccine, a live IBD vaccine and at least one vaccine selected from live vaccines against Newcastle disease virus (NDV), chicken infectious bronchitis virus (IBV), Marek's disease virus and chicken encephalomyelitis virus Can be combined. For inactivated vaccines,
IBD-inactivated vaccines and NDV, IBV, reduced oviposition syndrome 1976 virus, avian reovirus, bacteria (such as Escherichia coli and Salmonella), mycoplasmas (such as Mycoplasma galicepticum and Mycoplasma shinobier) and parasites (such as Eimeria). It can be combined with at least one vaccine selected from activated vaccines.

According to the present invention, there is provided an IBD vaccine capable of raising a neutralizing antibody against chicken IBD. According to the method of the present invention, an IBD vaccine of constant quality can be stably provided at low cost as compared with the conventional method. Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples.

[0014]

EXAMPLES Example 1 Eagle MEM medium (pH 7.2) containing 5% bovine serum 2
In 20 ml, 20 LMH cells (ATCC No. CRL-2117) were added.
Suspended to a density of 10,000 / ml, to which attenuated IBD
Virus-K strain (Yotsaburo Otaki. Chicken disease research bulletin Vol. 18
Supplement No., 1-15, 1982) was mixed to a moi 1 and the mixture was cultured in a roller bottle at 37 ° C. for 3 days with stirring. As a control, chicken embryo primary cells (CEF) were suspended in 200 ml of the above MEM medium to a density of 2,000,000 / ml, and attenuated IBD virus-K strain was added thereto.
i and mixed at 37 ° C. for 3 days while stirring the mixture in a roller bottle. These cultures were centrifuged at 1500 rpm, and the resulting supernatant was used as a virus suspension. When the virus content of the virus suspension was measured in accordance with the standard for biological preparations for animals (June 1993, published by the Japan Society for Animal Biology, pages 178 to 183), the results in Table 1 were obtained. Was.

[0015]

Table 1 ─────────────────────────────── Cell Virus content Virus production per cell (TCID ₅₀ / ml ) ( ₅₀ / 200,000 TCIDs) ─────────────────────────────── LMH 10 ^8.1 10 ^8.2 CEF 10 ^8.2 10 ^7.2 ───────────────────────────────

As is clear from the above results, the IBD virus culture method using LMH cells according to the present invention
Compared to the culture method, the amount of virus production per cell seeding density is about 10 times higher.

Example 2 A 14-day-old SPF chicken was administered drinking water with a live vaccine experimentally produced using the IBD virus propagated in LMH cells and a commercially available live vaccine produced using the IBD virus propagated in CEF. The neutralizing antibody titer after one week was measured in accordance with the standard for biological biological products for animals (June 1993, published by the Biological Society of Animals, pages 178 to 183). Was obtained.

[0017]

[Table 2] 中 和 Neutralizing antibody for vaccine cell test ─────── ───────────── Number of birds <200 200 800 ≧ 3200 ───────────────────────────── ─── Prototype LMH 100 046 Commercial product CEF 100 37 ─────────────────────────────── ─

As is evident from the above results, the live IBD vaccine obtained by growing in LMH cells according to the present invention,
They had sufficient titers for chickens as commercial products.

Claims (6)

[Claims] 1. A method for preparing an infectious bursal disease (IBD) virus, which comprises infecting a cell line derived from a bird with an infectious bursal disease virus and growing the virus. 2. The method for preparing an IBD virus according to claim 1, wherein the avian cell line is a chicken cell line. 3. The method for preparing an IBD virus according to claim 2, wherein the chicken cell line is an LMH cell. 4. An IBD vaccine comprising the IBD virus obtained by the preparation method according to claim 1 or an antigen component thereof. 5. A method for inhibiting the occurrence of IBD, wherein the method comprises administering the IBD vaccine according to claim 4 to poultry. 6. A combination vaccine comprising the IBD vaccine according to claim 4, and an antigen derived from at least one microorganism selected from another virus, mycoplasma, bacteria and parasites.