• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    The invention relates to a method of preparing an attenuated virus strain, which can be used to protect pigs, and in particular piglets, against transmissible gastroenteritis (TGE).
    公开号:SU971108A3
    申请号:SU782608003
    申请日:1978-04-21
    公开日:1982-10-30
    发明作者:Альберт Бахманн Петер；Майер Антон
    申请人:Н.В.Филипс Глоэлампенфабрикен(Фирма)；
    IPC主号:
    专利说明:

    one
    The invention relates to a method for obtaining a vagina, which can be used to protect the piglets from transmissible gastroenteritis.
    A method of obtaining a vaccine is known.
    dp protects pigs from transmissible gastroenteritis by repeatedly replanting the original virus to the culture of the kidney kpetok of a dog with a boarding school in 24 hours and less, and collecting the virus-containing Q of your material l.
    However, the known method is not sufficiently effective because it leads to limited protection of the pig from an infectious disease., 5
    The purpose of the invention is to increase the efficiency of the method.
    The delivered chain is achieved by 250-ZOO transfers of the original virus on the thyroid gland cell culture with it after 1-2 days while collecting the virus-containing material.
    It was found that if the vaccine obtained in accordance with the proposed method was administered orally to the sows, the smallpox virus continues to multiply along the intestinal tract of the animal. Pigs born from such sows are completely infected by the infection caused by the virulent transmissible gastroenteritis (TGE) virus with IgA antibodies (imgunoglobulin-A), kept in the colostrum.
    In tabl; Table 1 shows the titers of antibodies IgA of colostrum samples, determined in the course of testing with serum neutralization.
    Said samples are centrifuged, diluted with physiological NaCe solution (1:10) and heated to 4 ° C for 30% KfflH. Then the pH of the solution is adjusted to 4.6-4.65 using a mixture of equal volumes of 1N. NSV and 2M acetic acid, after which the resulting solution is stirred for 2O minutes. After centrifugation (12, OOOqj and 4 ° C), dialysis was performed overnight with respect to O, 1 M tris-hydroxymetamine, 0.2 M NaCC, the pH of which was up to 3.97 adjusted to 8.0 with 1 n HCE. Samples of this concentrate to half a volume of 5% CarbowdX (molecular weight 2O, LLC) and introduced into a column (2.5-100 cm) with SephcfdexG-200. Washout is carried out with Tris HACW buffer, pH 8.0. Table 1 ; IgA antibody titer After the TGE virus was introduced to the sows of the ZOO th subculture, repaired in accordance with the invention, a relatively high content of IgA antibodies was detected in the milk, Example 1. The initial virulent virus was isolated from the infected sow in Dnsti-tut Ur MikrobioEofepsis:) nfeciionsi (.pan1 tieHetlSeg in Munich. This exclusively pathogenic viral chain (hereinafter B1-chain) was isolated directly on living tissue of the thyroid gland. Cell cultures of living thyroid gland tissue were obtained using thyroid g glands obtained from the Munich slaughterhouse. The thyroid glands, after removing them from the packaging and sterilizing the surface, carried out twice with 95% alcohol, were cut into pieces 1-2 mm in size and washed three times with phosphate buffer (SRBF). or 0.37% trypsin buffer solution (not containing u), after which the drying was py fractionated with trypsin at 37 ° C. The first two trypsin fractions were decanted (after treatment for 1 h), and fractions 3 and 4 (processing time 1.5 h) was filtered and stored at temperatures e 4 ° C. At the end of this treatment, the cell trypsin suspension was centrifuged by E. E. Yumin (500 g). The liquid emerging on the surface was drunk off, and the precipitate was suspended in SRBP phosphate buffer 84 and centrifuged again. The cell pellet was placed in 10 ml of a 1 st medium. A solution of EigSe salt was used as the culture medium. However, other suitable culture media may also be used. The DGA culture medium contained 10% calf serum and 5% maintenance medium. In addition, the medium contained 5O ml of lactalbumin hydrolyzate per liter of medium, and possibly one or more antibiotics. Cell growth took place in conventional vessels for growing crops, for example, in flat glass or plastic vessels. The culture medium is replaced after 2-3 days. After surviving a culture for 4-6 days, the primary cell cultures were usually closed with a layer of cells. Secondary cell cultures obtained from the thyroid glands of the pig were used to pass through them B1-chains of the THE virus in the above-mentioned cells and to determine the infectious ability of the virus in various parts of the small intestine. The mentioned secondary cell cultures were obtained again by sowing primary cell cultures at a ratio of 1: 2. The virus was attenuated as a result of 250-350 consecutive passages on secondary cells of living tissues of the thyroid gland of pigs at 37 ° C. The successive transfers were carried out in the usual manner. Based on the pathogenic effect and using virus titrations, it was found that the virus titer is usually the maximum after incubation for 24-28 hours. Example 2. In order to determine the effect of successive passages on the soft tissue of the thyroid gland of pigs on the virulence of the virus, each 2 ml of virus-containing material after the 2nd (2x10 ECG / mp), 120th (2.10 ECG / mp), 250th (ETG / mlK 300th (4 x 10 EZG / ml) and 35O-th (4 U EZG / ml) subcultures. Titers are expressed in units of the hemolysis zone per ml (USH / ml.) At the age of 1–2 days, experimental animals were weaned from sows, which were taken with neutralizing antibodies that provided immunity against the TGE virus, and were watered from a bottle. On the second day of life, they were infected orally by injecting 2 ml of one of the above of the viruses containing the material, which occurred at a different number of transfers, ten control samples were given in 2 megapixels of the test medium. The course of the clinical course of burial was controversial twice a day. When the piglets remained alive on the tenth day, a blood sample was taken from them to determine the content of antibodies. The amount of antibodies was determined using a test based on serum neutralization using the microtiter method (Wilte K.H-i / Arcti.cyes Virusforset 1971, 33, S. 171-176). The first occurrence of diarrhea was considered as horses of the incubation period. Vomiting was observed only in those pigs that were injected with the virus after the second reseeding. For this reseeding, the incubation period was 16-18 hours. With a larger number of reseeding, an increase in the incubation period occurred, which reached 80 hours for the 300th reseeding. After being charged with viruses, the 2nd, 120th and 250th The reseed had diarrhea that lasted for several days. The pigs that received the virus after ZOOgo replanting differed in hard feces for several days, while the piglets that received the virus after 35O-GO replanting did not show any symptoms of the disease at all. The pigs that were infected with the virus after the second subculture, died i2 days later after the introduction of the virus. None of the six pigs that were injected with the virus after 12O-GO replanting remained alive. Of the four piglets that were injected with the virus of the 250th reseeding, only one survived. All animals infected with the virus obtained after the 250th reseeding recovered from the disease, as well as the pig that was injected with the virus after-25O reseeding. From these results it can be concluded that virulence up to the 250th reseeding was only slightly weakened, so that the disease caused by the infection turned out to be very serious, after which death occurred. Example 3. This experiment was carried out in order to determine where the reproduction of a virus obtained after a different number of passages of a virus weakened in accordance with the invention in the small intestine occurs. For this purpose, the virus was infused into newborn pigs after a different number of transfers, the same as in example 2 and by the same method. Immediately after the symptoms of typical infection with the TGE virus were noted, the porcine symptoms, which were held in isolation from each other and were infected individually, were killed. Beginning from the duodenum intestine, samples were taken with a total number of 10 units of the small intestine, taken at equal distances of 25-35 cm. These samples were used to determine the titer of the virus that is spread on various parts of the small intestine. The titer was determined after homogenizing the sample in a mortar and centrifuging a 10% suspension in SRBF. Ten times diluted samples were prepared from the emerging liquid, each time 0.2 ml of individual samples were sampled — 4 tubes and containing a pig cell culture of the pig. After incubation for 7 days at 37 ° C, a cytopogenic effect (CPE) was observed and the titer determination was carried out in accordance with the method of Kerber (lann: hn-5chm1edebercJ Lgs. Exp, Pdtlioe Phcirmoico, 162), 1931, r, 48O-482 ). - As follows from the results presented in the table above, the virulent virus obtained after the second reseeding is propagated throughout the small intestine but does not spread in it. This ® is the most relevant for viruses obtained after 120th and 25O-th transfers, For the 2nd case of reseeding, the virus titers are between 0.5 and 1, About TC ID 5O / O, 2 ml, while the titers for the virus after the 120th reseeding are between 2.0 and 4.5 Pocg ТС ID 5О / О, 2 ml. Similar titanium values. ditch were obtained for viruses after. 25O-GO and-ZOO-go transfers. In contrast, it can be shown that. the multiplication of the virus, obtained as a result of 35O transfers, occurs in the top. to the central part of the small intestine. PRI me R. 4. Experiments were performed with 13 pigs mi-females. At the beginning of the experiment, all pigs did not contain antibodies of the TGB virus. In group G consisting of 8 female pigs, immunity was established by double oral administration of viral material approximately 5 and 2 weeks before the expected farrowing. Immunity occurred upon oral administration of 4 ml of the viral material in an acid-resistant capule.
    7,9711088
    The troupe {(4 female pigs) has received a dp infestation of all piglets.
    half of the viral dose orally (2 ml) of 13 females on the third day after birth.
    in the capsule and half through the nose. Virus - For this purpose, 1 ml of the suspension was different material was harmless to pigs. It was compressed in such a way that it contained a control pig and was not 100-1OOO P f D (the infectious dose for receiving the virus. Pigs), and this dose was administered each as a virulent test species; rosenko. . Russa was taken Miller strain TGE virus. After three subcultures, a titer of neutralization of serum of pigs was prepared and a 10% finely suspended uterus suspension was prepared and grown after passive immunopted small intestine in PB5. After virusization, the TGEV1 chain is obtained by centrifuging, dividing into portions after ZOO th subculture, and 2 ml and freezing at a temperature of live pigs, 15 days after the re-70p, the indicated viral material of birth is given in Table. 2
    table 2
    10/10
    11/11
    9
    Group at 17
    29 33 35
    ; 971108
    10 I Continued gab, 2
    5/5
    7/6
    9/9
    权利要求:
    Claims (1)
    [1]
    SUMMARY OF THE INVENTION A method for producing a vaccine for protecting piglets from transmissible gastroenteritis by repeatedly reseeding the original virus on a tissue culture and collecting virus-containing material, characterized in that, with a chain for increasing the efficiency of the method, a pig thyroid cell culture is used as a tissue culture and 250-300 transfers are carried out after 1-2 days at. 37 ° C.
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    同族专利:
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    NL7704348A|NL7704348A|1977-04-21|1977-04-21|PROCEDURE FOR PREPARING A ATTENUATED TRANSMISSIBLE GASTROENTERITISVIRUS FOR USE IN LIVE VACCINES.|
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