METHOD OF PREPARING AN IMMUNOGEN OF PASTEURELLA MULTOCIDA

Abstract: The invention relates to a method of preparing an immunogen by cultivating a Pasteurella multocida strain for at most 12 to 24 hours in a liquid medium, removing the bacteria from the culture medium, filtering the culture filtrate, and rapidly freezing or freeze-drying this filtrate. The invention also relates to a method of preparing a vaccin against Pasteurella multocida infections, and to the vaccin per se.

Description: METHOD OF PREPARING AN IMMUNOGEN OF PASTEURELLA MULTOCIDA The invention relates to a method of preparing an immunogen by cultivating a Pasteurella multocida-strain and gaining the culture-filtrate. Such a method is known from the article by K.K. Shrivastava et al, Can. J. Microbiology, Vol. 23, (1977), 197 -201 In this article it is described that a culture-filtrate and a certain fraction thereof, respectively, provides some protection in mice when these are infected, after immunization, with 25 LD50 Pasteurella multocida bacteria. It has now been found that a very well active immunogen can be obtained by cultivating a Pasteurella multocida strain for 12-24 hours, preferably for 14-16 hours, in a liquid medium removing the bacteria from the culture medium, sterile filtering the culture filtrate and rapidly freezing or freeze-drying it, so as to avoid inactivation. It is essential for the strain to be cultivated for at most 24 hours, since in the case of longer-lasting cultivation, the immunogen y does not increase or increases hardly, or even decreases, while the toxicity of the culture filtrate in the case of longer cultivation increases rapidly in particular when toxin-producing strains are used. The cultivation of the strain can be carried out in a manner known per se in a liquid cultivation medium suitable for this purpose at 37 0C. A suitable cultivation medium is, for example, brain-heart-infusion medium (BHI -medium). It has been found that the immunogen in the culture filtrate to be obtained according to the above method is very sensitive to both chemical and thermal inactivation. When the liquid culture filtrate is kept at 600C for 30 minutes, or at +4 0C for a longer period of time, or kept frozen at -200C, the immunogenicity of the composition thus treated decreases considerably. The immunogenic activity also decreases by a treatment with 0.3% formalin for 24 hours or with thimerosal (1 : 10,000). Various Pasteurella multocida strains may be used as starting materials. For example, good results are obtained by cultivating in the above-described manner, and further treating as starting materials, strains isolated from pigs (for examples the strain of the Carter D-type which is known under No. 47459-449 at the Centraal Diergeneeskundig Instituut (CDI) (Central Veterivary Institute), or the strain of the Carter type A which is known under No. 53566 at the CDI), or a strain isolated from turkeys (for example, the strain 3 USA, standard type 3, P-1059). The following properties of the immunogen to be obtained according to the invention may be mentioned: a) in the wet state the immunogen is very thermolabile, i.e. sensitive to temperature; b) in the freeiidried state the immunogen is comparatively insensitive to temperature (i.e. stable); c) the immunogen is sensitive to chemical inactivation with, for example, formalin or thimerosal; d) the immunogen to be obtained according to the invention may be used in known manner in vaccins for the control of infections caused by Pasteurella multocida, for example, atrophic rhinitis in pgis, fowl cholera in poultry, haemorrhagic septicaemia in cattle and pasteurellosis c.q. pasteurella infections in sheep, cattle, pigs, rabbits and man; e) the immunogen may be used for the preparation of combina tion vaccins, for example, in a vaccin against atrophic rhinitis it may be combined with Bordetella bronchiseptica antigen; f) by the addition of adjuvants the protective action can be even further increased. The invention will now be described in greater detail with reference to the following examples in which the immunogenic activity of the culture filtrate obtained is demonstrated both in direct and in indirect protection tests. EXAMPLE I A freeze-dried inoculation culture of a Pasteurella multocida strain of the Carter type D (known at the CDI under No. 47459-449) isolated from pigs was used as a starting material for the preparation of the immunogen. The contents of an inoculation ampoule, after reconstitution in distilled water, were spread on sheep blood-agar medium and incubated at 3700. After 16 hours the separate colonies were transferred to a starting culture of BHI-medium. After 8 hours' incubation at 370C on a shaking machine (80 rpm), inoculation was again carried out (5% vol/vol) on 3H1 medium for the production culture. Incubation was carried out at 370C for 16 hours on a shaking machine (80 rpm). The cultivated Pasteurella germs were then centrifuged at 10000 rpm (17,000 x g) for 30 minutes. The supernatant was decanted and filtered sterile; the culture filtrate thus obtained was frozen at -700C. EXAMPLE II Dilutions indicated in table A were prepared from the culture filtrate (CF) obtained according to Example I. Per dilution, 10 mice were vaccinated once intraperitoneally (i.p.) with 0.5 ml. Two weeks after vaccination the animals were challenged i.p. with 104LD50 germs of the homologous strain. The percentage of surviving animals 1 week after challenge is recorded in Table A. TABLE A EMI3.1 <tb> CF-dilution <SEP> % <SEP> survivors <tb> <SEP> 1 <SEP> : <SEP> 10 <SEP> 90 <tb> <SEP> 1 <SEP> : <SEP> 102 <SEP> 100 <tb> <SEP> 1 <SEP> : <SEP> 103 <SEP> 90 <tb> <SEP> 1 <SEP> : <SEP> 104 <SEP> 100 <tb> <SEP> 1 <SEP> : <SEP> 105 <SEP> 30 <tb> <SEP> : <SEP> leo6 <SEP> 40 <SEP> <tb> <SEP> 1 <SEP> : <SEP> 107 <SEP> O <SEP> <tb> Control <SEP> group <SEP> 0 <tb> Summarizing it may be concluded that mice, after one vaccination with 10,000 x diluted culture filtrate of the strain mentioned in Example I, are protected for 100X against a challenge infection with 104 LD50 germs of the homologous strain. EXAMPLE III In the manner described in Example I, a culture filtrate was prepared starting from the pig's strain of the Carter type A (known at CDI under no. 53566). The dilutions recorded in Table B were made from this culture filtrate. Per dilution, 10 mice were vaccinated i.p. with 0.5 ml. This was repeated after a fortnight. Two weeks after the last vaccination the animals were challenged i.p. with the quantity of germs of the homologous Pasteurella multocida A strain mentioned in Table B. One week after challenge the percentage of animals recorded in Table B was still alive. TABLE B EMI4.1 <tb> CF-dilution <SEP> challenge <SEP> % <SEP> survivors <tb> <SEP> dose <SEP> .... <SEP> <SEP> LD <SEP> 0 <SEP> <tb> <SEP> 1:2 <SEP> 50 <SEP> 100 <tb> <SEP> 1:4 <SEP> 50 <SEP> 100 <tb> <SEP> 1:8 <SEP> 500 <SEP> 100 <tb> <SEP> 1 <SEP> : <SEP> 16 <SEP> 500 <SEP> 100 <tb> <SEP> 1 <SEP> : <SEP> 32 <SEP> 500 <SEP> 1 <SEP> 80 <tb> <SEP> 1 <SEP> : <SEP> 64 <SEP> 5,000 <SEP> 100 <tb> <SEP> 1 <SEP> : <SEP> 128 <SEP> 5,000 <SEP> 100 <tb> <SEP> 1 <SEP> 1 <SEP> : <SEP> 256 <SEP> 50,000 <SEP> 100 <tb> <SEP> 1 <SEP> : <SEP> 512 <SEP> 501000 <SEP> 0 <tb> Control <SEP> group <SEP> 50 <SEP> 0 <tb> Summarizing it may be concluded that mice after two vaccinations with 256 x diluted culture filtrate of the said strain are protected for 100% against a challenge infection with 5 x 104 LD50 germs of the homologous strain. EXAMPLE IV With the culture filtrate (CF) obtained in Example I various vaccins were formulated: 1) undiluted CF without an adjuvant 2) undiluted CF with adjuvant A 3) freeze-dried CF with adjuvant A 4) freeze-dried CF combined with Bordetella bronchiseptica vaccin 5) freeze-dried CF with adjuvant B 6) freeze-dried CF with adjuvant C. Per formulation four pigs were vaccinated twice i.p. (on day 0 and on day 14) with 2 ml of the formulation. Each dose contained the same quantity of immunogen. Two weeks after the last vaccination blood was drawn and serum gained herefrom. The sera thus obtained were tested for the presence of protective antibodies, in a passive protection test in mice. Of each group of pigs, vaccinated with one of the vaccin formulations, a mixed serum was made and 0.5 ml hereof per mouse were administered içp. to groups of 10 mice. The next day the mice were challenged i.p. with 20 LD50 germs of the homologous Pasteurella multocida strain D (see Example I) in 0.5 ml. One week after challenge the percentages of mice recorded in Table C were still alive. TABLE C EMI5.1 <tb> vaccin <SEP> formulation <SEP> % <SEP> protection <tb> <SEP> 1 <SEP> 20 <tb> <SEP> 2 <SEP> 100 <tb> <SEP> 3 <SEP> 90 <tb> <SEP> 4 <SEP> 70 <tb> <SEP> 5 <SEP> 100 <tb> <SEP> 6 <SEP> 80 <tb> <SEP> Control <SEP> group <SEP> ¯¯¯ <SEP> <SEP> 20 <tb> Summarizing it may be concluded that in sera of pigs which have been vaccinated twice with immunogen, combined with adjuvant, protective antibodies can be demonstrated. Adjuvation of the immunogen increases the humoral immune response. EXAMPLE V In the manner described in Example I, a culture filtrate (CF) was prepared from material of the Pasteurella multocida strain 3 USA (a turkey strain known under No. P-1059). The dilutions mentioned in Table D were made from this CF. With each dilution, 3 mice were vaccinated twice (day 0 and day 14) i.p. with 0.5 ml. Two weeks after the last vaccination the animals were challenged i.p. with the quantity of bacteria of the homologous Pasteurella multocida strain 3 USA recorded in Table D. One week after challenge the percentage of animals mentioned in Table D were still alive. TABLE D EMI6.1 <tb> CF <SEP> - <SEP> dilution <SEP> Infection <SEP> % <SEP> Survival <tb> <SEP> dose <SEP> ..... <SEP> LD50 <tb> <SEP> 1 <SEP> : <SEP> 2 <SEP> 50 <SEP> 67 <tb> <SEP> 1 <SEP> : <SEP> 4 <SEP> 50 <SEP> 100 <tb> <SEP> 1 <SEP> : <SEP> 8 <SEP> 500 <SEP> 100 <tb> <SEP> 1 <SEP> : <SEP> 16 <SEP> 500 <SEP> 100 <tb> <SEP> 1 <SEP> : <SEP> 32 <SEP> 5,000 <SEP> 100 <tb> <SEP> 1 <SEP> : <SEP> 64 <SEP> 5,000 <SEP> 100 <tb> <SEP> 1 <SEP> : <SEP> 128 <SEP> 50,000 <SEP> 100 <tb> <SEP> 1 <SEP> : <SEP> 256 <SEP> 50,000 <SEP> 100 <tb> <SEP> 1 <SEP> : <SEP> 512 <SEP> 500,000 <SEP> 0 <tb> <SEP> 1 <SEP> : <SEP> 1024 <SEP> 500,000 <SEP> 33 <tb> Control <SEP> group <SEP> 50 <SEP> 0 <tb> Summarizing it may be concluded that mice, after two vaccinations with 256 x diluted culture filtrate of the said strain, are protected for 100% against a challenge infection with 5 x 10 LD50 germs of the homologous strain.

Claims: CLAIMS: 1. A method of preparing an immunogen by cultivating a Pasteurella multocida strain, and gaining the culture filtrate, characterized in that a Pasteurella multocida strain is cultivated for at most 12-24 hours in a liquid medium, the bacteria are removed from the culture liquid, the culture filtrate is filtered sterile and is rapidly frozen or freeze -dried so as to avoid inactivation. 2. A method as claimed in Claim 1, characterized in that, after cultivating for 14-16 hours, the bacteria are separated by centrifuging or ultrafiltration. 3. A method of preparing a vaccin against Pasteurella multocida infections, characterized in that an immunogen obtained according to Claim 1 or 2 is brought into a form suitable for administration. 4. A method as claimed in Claim 3, characterized in that an adjuvant is added. 5. A method as claimed in Claim 3 or 4, characterized in that one or more other active components are added. 6. A method as claimed in Claims 3-5, characterized in that a vaccin is prepared against atrophic rhinitis. 7. A method as claimed in Claim 6, characterized in that Bordetella bronchiseptica antigen is incorporated in the vaccin. 8. A vaccin obtained according to the method as claimed in Claims 3-7.

Inventors: DE WACHTER, JAN CORNELIS, PRANGER, MAARTEN HENDRIK
Applicants: DUPHAR INTERNATIONAL RESEARCH B.V
Application year: 1983
Main classification: A61K39
IPC codes: A61K39