METHOD OF IMMUNIZING PIGS AGAINST AUJESZKY'S DISEASE

Abstract: The present invention relates to a method of immunizing pigs against Aujeszky's disease by intranasal administration of a living attenuated Aujeszky virus vaccine, to a method of preparing a vaccine suitable for this purpose, and to the so obtained vaccine. The vaccine can be used to protect sero-positive piglets.

Description: METHOD OF IMU}JIZI!G PIGS AGAINST AUJESZKYsS DISEASE. The invention relates to a method of immunizing pigs against Aujeszky's disease by intranasal administration of a living attenuated Aujeszky-virus vaccine, to a method of preparing a vaccine suitable for this purpose, and to the vaccine thus obtained. Aujeszky's disease or pseudorabies is a generally occurring infection in many kinds of ani:nals. In particular in pigs it is often an acute cause of death and the disease causes enormous economic losses. In pregnant animals, Aujeszkyts disease is characterized by abortipn and stillbirth. In newly born pigs the disease causes a high mortality as a result of nervous affections. In breeding-pigs, later litters general suffer little from the disease because the animals are protected during the most vulnerable period of their lives against the virus by maternal antibodies via the colostrum of latent infected sows. On the other hand, Aujeszky's disease in the intensive pig-fattening system where piglets originating fro different places are usually put together, is an ever returning problem. Outbreaks of pseudorabies in piglets after the lactation period make this disease in the intensive pig -fattening to be one of the most important diseases of swine. In order to protect piglets against Aujeszky's disease, a few good injection vaccines are available. J.B. McFerran and C. Dow, for example, in Res. Vet. Sci. (1975), 19, 17-22 describe that sero-negative pi-- lets, that is to say piglets without maternal antibodies, can well be protected against subsequent challenge with virulent virus both by means of intramuscular injection and by intranasal administration of an Aujeszky virus martha strain or K-strain) grown in Vero cells. McFerran et al, however, come to the conclusion that intranasal inocu'-t cn with the K-strain proves to be a disappointment. Since Aujeszky virus first of all multiplies in the upper part of the respiratory tract, it was expected that the local immunity after intranasal administration would be stimulated and in this manner colon isat ion of the upper respiratory tract by pathogenic strains would be prevented. Although a slight decrease in the degree of clinical symptoms following challenge with virulent virus could be observed after intranasal administration, these minimum advantages did not outweigh the disadvantages associated with an intranasal administration. Good results are furthermore known, obtained by Howarth (Proc. 74th Ann. Meet. U.S. Animal Health Ass. (1972), 371-384) after intranasal administration of Aujeszky virus of the BUK-strain passaged in porcine kidney cell line. These experiments were also carried out with animals which had repeatedly bee found to be sero-negative. The disadvantage of these injection vaccines, which give good results i sero-negative animals, is that they are not active in sero-positive piglets as long as they are maternally immune. This problem particularly occurs in areas where one strives after a hngh titer of maternal antibodies in young animals through a good vaccination scheme of the breeding sows. It has surprisingly been found now that piglets can be very successfully vaccinated against Aujesz'xy's disease already early, i.e. at an age at which they usually are still maternally immune, by vaccinating the intranasally with a living attenuated Aujeszky virus vaccine which has been pass aged in mammal cells or ir/a continuous cell line on the basis of mammal cells. A vaccine which is suitabLe for use in the method according to the Invention is, for example, a vaccine which is obtained by cultivating virus of the Bartha strain (K -strain) in mammal cells or in a continuous cell line on the basis of mammal cells. An extremely suitable vaccine according to the invention is obtained, for example, by cultivating virus of the Bartha strain in secondary porcine kidney cells or in porcine kidney continuous cell line. It has furthermore been found that after intranasal administration of a vaccine according to the invention; a very gradual build-up of immunity takes place. In fattening pigs which are usually slaughtered at the age of approximately 26 weeks, the antibody titre goes on increasing up to the moment of slaughtering when the animals are vaccinated with a quantity of vaccine which contains 10 - 107 TCID50, pre ferably approximately 1C6 TOlD50 of virus. In this manner the animals can be optimally protected against the disease and economic losses as a result of the disease can be minimized. Although an early immunization and gradual build-up of immunity by means of the vaccine according to the invention is important in the first place in intensive pig-fattening systems, it was found that the new vaccine is also of great importance for the protection of breeding animals. It appeared that a very good and long standing sensibilisation of the immune system is obtained after one intranasal administration of the vaccine according to the invention. This means that the defence system in animals which have beemade sensible in this way can be activated in an unprecedented degree by giving them an injection with the vaccine (booster-effect). The invention will now be described in greater detail with reference to the ensuing experiments. EXPERIMENT I In this experiment, 23 piglets of 9 weeks old were used. Ten of these piglets were sero-positive (maternally immune) and the remaining 13 were sero-negative. The animals were vaccinated intranasally with 106 TCID50 of Aujeszky virus which had been cultivated in chicken embryo fibroblasts (6 sero-negative and 4 sero-positive animals), or in a continuous cell line (porcine kidney originating from the institute of Prof. Pensaert), (7 sero-negative and 6 sero-posi tive animals). The results, i.e. serum neutralization tltres, obtained with the two vaccines in the various groups are recorded in Tables A (sero-negative animals) ar.c R (sero -positive animals) Stated in Tables C and D for these groups of animals is the variation of the number of sero-negative animals at a few points of time after inoculatIon with the two vaccines. From Tables A to D it appears that a vaccine pro duced on cell line gives a clear serologic response in sero -positive animals after one intranasal administration. Vaccine produced in chicken embryo fibroblasts (CEF) gives a much smaller serologic response. In sero-positive animals it is only prevented that the titre drops to zero. TABLE A Sero-negatice animals: EMI4.1 <tb> <SEP> before <SEP> after <tb> <SEP> vaccination <SEP> vaccination <tb> time <SEP> in <SEP> weeks <SEP> 0 <SEP> # <SEP> 2 <SEP> 6 <SEP> 10 <SEP> 14 <tb> <SEP> CEF <SEP> vaccine <SEP> 0 <SEP> # <SEP> 1,8 <SEP> 2,5 <SEP> - <SEP> 2,9 <SEP> 2,6 <tb> <SEP> Cell <SEP> line <SEP> vaccine <SEP> 0 <SEP> 2,2 <SEP> 4,7 <SEP> 6,8 <SEP> 5,8 <tb> TABLE B Sero-positive animals: : EMI4.2 <tb> <SEP> before <SEP> after <tb> <SEP> vaccination <SEP> vaccination <tb> time <SEP> in <SEP> weeks <SEP> 0 <SEP> 2 <SEP> 6 <SEP> 10 <SEP> 14 <tb> CEF <SEP> vaccine <SEP> 2,0 <SEP> 3,0 <SEP> 1,3 <SEP> 1,4 <SEP> 1,5 <tb> Cell <SEP> lir.e <SEP> vaccine <SEP> 2,0 <SEP> 2,4 <SEP> - <SEP> 2,7 <SEP> 4,9 <SEP> 5,8 <tb> TABLS C Variation of the number of sero-negative animals in te group which was sero-negative before vaccination: : EMI4.3 <tb> <SEP> before <SEP> after <tb> <SEP> vaccination <SEP> vaccination <SEP> <tb> time <SEP> in <SEP> weeks <SEP> 0 <SEP> 2 <SEP> 6 <SEP> 10 <SEP> 14 <SEP> t4 <SEP> <tb> CEF <SEP> vaccine <SEP> 6 <SEP> 3 <SEP> 3 <SEP> 3 <SEP> 3 <tb> Cell <SEP> line <SEP> vaccine <SEP> 7 <SEP> 1 <SEP> 1 <SEP> 1 <tb> TABLE D Variation of the number of sero-negative animals in the grou which was sero-positive before vaccination: : EMI4.4 <tb> <SEP> before <SEP> after <tb> <SEP> vaccination <SEP> vaccination <tb> time <SEP> in <SEP> weeks <SEP> 0 <SEP> 2 <SEP> 6 <SEP> 10 <SEP> 14 <tb> CEF <SEP> vaccine <SEP> 0 <SEP> 0 <SEP> 3 <SEP> 3 <SEP> 3 <tb> Cell <SEP> line <SEP> vaccine <SEP> 1 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <tb> EXPERIMENT II Ten piglets from a group of 39 ten weeks old sero-positive piglets (i.e. with maternal antibodies from the colostrum) were used as non-vaccinated control animals, 14 were vaccinated intranasally with 10 TCID50 of a vaccine (Bartha strain) cultivated in C.E.F., arrd the remaining 15 were vaccinated intranasally with 106 TCID50 of a vaccine (Bartha strain) cultivated in cell line (porcine kidney). In Tables E and F are recorded the serum neutralization titres at various instants and the variation of the number of sero-negative animals in the three sub-groups, respectively. As in Experiment r, it appears clearly that the vaccine cultivated in cell line is much more effective than the vaccine cultivated in C.E.F. TABLE E Sero-Dositive animals: EMI5.1 <SEP> before <SEP> after <tb> <SEP> vaccination <SEP> vaccination <tb> time <SEP> in <SEP> weeks <SEP> 3 <SEP> 0 <SEP> 2 <SEP> 4 <SEP> 8 <SEP> 12 <SEP> 15 <tb> Not <SEP> vaccinated <SEP> 1,6 <SEP> 1,0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <tb> CEF <SEP> vaccine <SEP> 1,9 <SEP> not <SEP> det. <SEP> 1,2 <SEP> 1,1 <SEP> 1,3 <SEP> 1,3 <SEP> 1,5 <tb> Cell <SEP> Line <SEP> vaccine <SEP> 1,7 <SEP> not <SEP> det. <SEP> 1,9 <SEP> 1,4 <SEP> 2,1 <SEP> 2,6 <SEP> 4,4 <tb> TABLE F Variation of the number of sero-negative animals in the group which was sero-positive before vaccination: EMI5.2 <tb> <SEP> Before <SEP> rafter <SEP> <tb> <SEP> vaccination <SEP> vaccination <tb> time <SEP> in <SEP> weeks <SEP> 3 <SEP> 0 <SEP> 2 <SEP> 4 <SEP> 8 <SEP> 12 <SEP> <SEP> 15 <SEP> <tb> Not <SEP> vaccinated <SEP> 2 <SEP> 6 <SEP> 8 <SEP> 10 <SEP> 10 <SEP> 10 <SEP> 10 <tb> CEF <SEP> vaccine <SEP> 3 <SEP> not <SEP> det. <SEP> 7 <SEP> 7 <SEP> 9 <SEP> 10 <SEP> 10 <tb> Cell <SEP> Line <SEP> vaccine <SEP> 2 <SEP> not <SEP> det. <SEP> 0 <SEP> O <SEP> 2 <SEP> 3 <SEP> 1 <SEP> O <SEP> <tb> The serum neutralization titres recorded in Tables A, B and E are stated as a serum dilution which still gives 50% neutralization of the living virus used in the experiment (100 TCID50). EXPERIMENT III A group of 39 sero-positive piglets were used (12 weeks old). Nine animals served as non-vaccinated control animals. Ten animals were vaccinated intramuscularly with 105 TCID50 vaccine cultivated in C.E.F. (Bartha strain). Ten animals were vaccinated intramuscularly 'with 106 TCID50 vaccine cultivated in (porcine kidney) cell line (Bartha strain). Ten animals were vaccinated intranasally with 106 TCID50 vaccine cultivated in (porcine kidney) cell line (Bartha strain) The serum neutralization titres at various instants before and after vaccination are recorded in Table G. From this it appears that after one intranasal vaccination with vaccine cultivated in cell line, the quantity of antibodies in maternal immune piglets increases considerably. This in contrast with the titres obtained < ser one intramuscular vaccination with a vaccine cultivated in C.E.F. and a vaccine cultivated in cell line. TABLE G Serum neutralization titres: EMI6.1 <tb> <SEP> Before <SEP> after <tb> <SEP> vaccination <SEP> vaccination <tb> time <SEP> in <SEP> weeks <SEP> 9 <SEP> 0 <SEP> 4 <SEP> 8 <SEP> 12 <SEP> 15 <tb> Not <SEP> vaccinated <SEP> 5,7 <SEP> 1,4 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <tb> 1 <SEP> x <SEP> CEF <SEP> vaccine <SEP> 6,9 <SEP> 1,3 <SEP> 0,1 <SEP> 1,4 <SEP> 2,4 <SEP> 2,1 <tb> <SEP> (intramusc.) <tb> 1 <SEP> x <SEP> Cell <SEP> Line <SEP> vacc. <SEP> 4,9 <SEP> 0,1 <SEP> 6,1 <SEP> 2,2 <SEP> 2,0 <SEP> 2,2 <tb> <SEP> (intramusc.) <tb> 1 <SEP> x <SEP> Cell <SEP> Line <SEP> vacc. <SEP> 5,9 <SEP> - <SEP> 1,3 <SEP> # <SEP> 1,9 <SEP> 4,8 <SEP> 6,7 <SEP> 6,7 <tb> <SEP> (ir.ranas.) <SEP> ¯ <SEP> <tb> EXPERIMENT IV Frcm a group of piglets a number were used as non-vaccinated control animals, another part was vaccinated once intra muscularly with 105 TCID50 vaccine cultivated in C.E.F., and a third part of the group was vaccinated once intranasally with 106 TCID50 vaccine cultivated in cell line in the same manner as in Experiment III. A number of animals from each of the three sub -groups were challenged intranasally 7 weeks after the vacci- nation with 105 TCID50 of a virulent Aujeszky strain (NIA-3). Another part of the three sub-groups was challenged in the same manner 15 weeks after vaccination. The number of days of growth retardation in the various groups is recorded in Table H. Table J shows the variation of the body temperature (in C) from 3 days before till 10 days after intranasal challenge. TABLE H Number of days of growth retardation after challenge EMI7.1 <tb> <SEP> Challenge <SEP> Challenge <tb> <SEP> 7 <SEP> weeks <SEP> 15 <SEP> weeks <tb> <SEP> after <SEP> vacc. <SEP> after <SEP> vaccination. <tb> Non-vaccination <SEP> <SEP> 12 <SEP> days <SEP> 19 <SEP> days <SEP> <tb> control <SEP> animals <tb> 1 <SEP> x <SEP> Intramuscular <SEP> <SEP> 7 <SEP> days <SEP> 13,3 <SEP> days <SEP> <tb> CEF <SEP> vaccine <tb> 1 <SEP> x <SEP> Intranasal <tb> cell <SEP> line <SEP> vaccine <SEP> 0 <SEP> days <SEP> 6,7 <SEP> days <tb> TABLE J Variation body temperature after challenge infection Challenge 7 weeks after vaccination day EMI8.1 <SEP> -3 <SEP> -2 <SEP> -1 <SEP> 0 <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9 <SEP> 10 <tb> non-vaccinated <tb> control <SEP> animals <SEP> 39.3 <SEP> 39.3 <SEP> 39.2 <SEP> 39.3 <SEP> 39.3 <SEP> 39.8 <SEP> 41.0 <SEP> 40.6 <SEP> 40.9 <SEP> 40.7 <SEP> 40.7 <SEP> 39.7 <SEP> 39.6 <SEP> 39.3 <tb> 1 <SEP> x <SEP> Intramuscular <tb> CEF <SEP> vaccine <SEP> 39.5 <SEP> 39.5 <SEP> 39.5 <SEP> 39.6 <SEP> 39.5 <SEP> 40.0 <SEP> 40.8 <SEP> 40.6 <SEP> 40.6 <SEP> 39.5 <SEP> 39.5 <SEP> 39.5 <SEP> 39.4 <SEP> 39.3 <tb> 1 <SEP> x <SEP> Intranasal <tb> cell <SEP> line <SEP> vaccine <SEP> 39.4 <SEP> 39.4 <SEP> 39.4 <SEP> 39.4 <SEP> 39.4 <SEP> 39.4 <SEP> 39.8 <SEP> 39.9 <SEP> 39.6 <SEP> 39.5 <SEP> 39.3 <SEP> 39.4 <SEP> 39.4 <SEP> 39.2 <tb> Challenge 15 weeks after vaccination EMI8.2 <SEP> -3 <SEP> -2 <SEP> -1 <SEP> 0 <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9 <SEP> 10 <tb> non-vaccinated <tb> control <SEP> animals <SEP> 39.5 <SEP> 39.6 <SEP> 39.6 <SEP> 39.5 <SEP> 39.7 <SEP> 40.0 <SEP> 40.8 <SEP> 41.0 <SEP> 40.7 <SEP> 40.8 <SEP> 40.3 <SEP> 40.1 <SEP> 39.8 <SEP> 39.7 <tb> 1 <SEP> x <SEP> Intramuscular <tb> CEF <SEP> vaccine <SEP> 39.3 <SEP> 39.5 <SEP> 39.7 <SEP> 39.9 <SEP> 39.8 <SEP> 40.2 <SEP> 40.6 <SEP> 40.6 <SEP> 40.4 <SEP> 40.4 <SEP> 40.0 <SEP> 39.3 <SEP> 39.7 <SEP> 39.6 <tb> 1 <SEP> x <SEP> Intranasal <tb> cell <SEP> line <SEP> vaccine <SEP> 39.3 <SEP> 39.3 <SEP> 39.3 <SEP> 39.3 <SEP> 39.4 <SEP> 39.4 <SEP> 39.6 <SEP> 40.2 <SEP> 40.4 <SEP> 39.6 <SEP> 39.3 <SEP> 39.4 <SEP> 39.3 <SEP> 39.4 <tb> EXPERIMENT V A group of 7 nine weeks old sero-positive piglets was vaccinated intranasally with a dose of 106 TCID50 of a live attenuated vaccine (Bartha strain) cultivated in porcine kidney cell line. On the age of 34 weeks 3 of the piglets (nos 1, 2 and 3) were vaccinated again with 105.5 5 TCID50 of the same vaccine, however, this time by means of an intramuscular injection. One other piglet (no. 4) was intramuscularly vaccinated with 104.0 TCID50 of the same vaccine. The 3 remaining piglets (nos. 5, 6 and 7) were injected with 108.2 TCID50 of inactivated vaccine. The results, i;e. the serum neutralization titers of the seven piglets are recorded in table K. The booster -effect appears clearly from these figures. TABLE K Titer at the age of .... weeks ¯¯¯¯¯¯¯¯¯¯¯¯¯ EMI9.1 <tb> Piglet <SEP> no <SEP> 9 <SEP> 11 <SEP> 13 <SEP> 17 <SEP> 21 <SEP> 26 <tb> <SEP> 1 <SEP> 3 <SEP> 8 <SEP> 12 <SEP> 45 <SEP> 32 <SEP> 45 <tb> <SEP> 2 <SEP> 4 <SEP> 22 <SEP> 89 <SEP> 64 <SEP> 64 <SEP> 45 <tb> <SEP> 3 <SEP> 8 <SEP> 16 <SEP> 32 <SEP> 89 <SEP> 64 <SEP> 89 <tb> <SEP> 3 <SEP> 8 <SEP> 16 <SEP> 32 <SEP> 89 <SEP> 64 <SEP> 89 <SEP> 45 <tb> <SEP> 4 <SEP> 6 <SEP> | <SEP> 545 <SEP> )178 <SEP> 128 <SEP> )178 <SEP> / <SEP> 89 <SEP> 64 <tb> <SEP> 5 <SEP> 16 <SEP> 12 <SEP> 64 <SEP> 64 <SEP> 45 <SEP> 89 <SEP> 45 <tb> <SEP> 6 <SEP> 8 <SEP> 32 <SEP> 128 <SEP> 128 <SEP> 128 <SEP> 256 <SEP> 12R <tb> <SEP> 7 <SEP> 3 <SEP> )45 <SEP> 12 <SEP> 64 <SEP> 12 <SEP> 32 <SEP> 45, <tb> EMI9.2 piglet <SEP> no. <SEP> 34 <SEP> wks <SEP> and-/ <SEP> 35 <SEP> 1 <SEP> 36 <SEP> 38 <SEP> 42 <SEP> 5 <SEP> "9 <tb> <SEP> 3 <SEP> days <tb> <SEP> 1 <SEP> 120 <SEP> 890 <SEP> 1780 <SEP> 450 <SEP> 220 <SEP> 128 <SEP> I <tb> <SEP> 2 <SEP> 450 <SEP> 2560 <SEP> 1780 <SEP> 1280 <SEP> 640 <SEP> 17 <SEP> ;"t! <tb> <SEP> 3 <SEP> 60 <SEP> 25j <tb> <SEP> 3 <SEP> 60 <SEP> 1780 <SEP> 3550 <SEP> 1780 <SEP> 220 <tb> <SEP> 4 <SEP> 450 <SEP> 2560 <SEP> 7080 <SEP> 5120 <SEP> 640 <SEP> 1024 <SEP> 12 <tb> <SEP> 5 <SEP> 120 <SEP> 2560 <SEP> 3550 <SEP> 2560 <SEP> 640 <SEP> 178 <SEP> 256 <tb> <SEP> 6 <SEP> 320 <SEP> 1780 <SEP> 2560 <SEP> 890 <SEP> 450 <SEP> 89 <SEP> 3-9 <tb> <SEP> 7 <SEP> 220 <SEP> 640 <SEP> 890 <SEP> 320 <SEP> 160 <SEP> 128 <SEP> 18 <tb>

Claims: CLAIMS: 1. A method of immunizing pigs against Aujeszky's disease by intranasal administration of a living attenuated Aujeszky virus vaccine, characterized in that maternally immune animals are inoculated with a virus cultivated in mammal cells or in a continuous cell line on the basis of mammal cells. 2. A method as claimed in Claim 1, characterized in that virus from the Bartha strain is used. 3. A method as claimed in Claim 1 or 2, characterized in that the virus is cultivated in secondary porcine kidney cells or in porcine kidney continuous cell line. 4. A method as claimed in Claims 1-3, characterized in that 10 - 107 TCID50 is administered. 5. A method as claimed in Claims 1-4, characte rlzed in that 106 TCID50 is administered. 6. A method as claimed in. Claims 1-5, characterized in that the intranasal inoculation is followed by an intramuscular booster injection. 7. A method of preparing an Aujeszky virus vaccine suitable for use in the method as claimed in Claims 1-6, characterized in that Aujeszky virus is cultivated in mammal cells cr in a continuous cell line on the basis of mammal cells and the resulting virus suspension is brought at the desired titre. 8. A vaccine obtained according to the method as claimed in Claim 7.

Inventors: ZANTINGA, JAN WILLEM
Applicants: DUPHAR INTERNATIONAL RESEARCH B.V
Application year: 1982
Main classification: A61K39
IPC codes: A61K39