WO1980002113A1 - Vaccin pour lutter contre la pleuropneumonie chez le cochon procede et substrat pour la fermentation aerobie hemophilus pleuropneumoniae - Google Patents

Vaccin pour lutter contre la pleuropneumonie chez le cochon procede et substrat pour la fermentation aerobie hemophilus pleuropneumoniae Download PDF

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WO1980002113A1
WO1980002113A1 PCT/DK1980/000021 DK8000021W WO8002113A1 WO 1980002113 A1 WO1980002113 A1 WO 1980002113A1 DK 8000021 W DK8000021 W DK 8000021W WO 8002113 A1 WO8002113 A1 WO 8002113A1
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pleuropneumoniae
vaccine
fermentation
process according
fermenting
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PCT/DK1980/000021
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English (en)
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J Fogh
H Riising
B Nielsen
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Nordisk Droge & Kemikalie As
J Fogh
H Riising
B Nielsen
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Priority to DE19803041448 priority Critical patent/DE3041448A1/de
Publication of WO1980002113A1 publication Critical patent/WO1980002113A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55588—Adjuvants of undefined constitution
    • A61K2039/55594—Adjuvants of undefined constitution from bacteria

Definitions

  • Haimophilus-like bacteria were isolated in Great Britain, California and Argentina from herds of pigs which were attacked by pleuropneumonia. It was the bacterium Haemophilus parahaemolyticus - or Haemophilus pleuropneumoniae as the Argentine strain was called - which was the cause of the disease pleuropneumonia. Since then attacks of the disease have been reported in many countries, including Denmark, cfr. R. Nielsen, Nord. Vet. Med. 22 (1970, 240-245.
  • the disease has a very acute course and it often accompanied by a high, mortality and since it appears rather frequently in many herds of pigs, some efforts have been made in an attempt to develop a vaccine which would protect the pigs against the disease.
  • a common feature of these vaccines is their use of killed cells of H. pleuropneumoniae and in most cases also an addition of an adjuvant.
  • Vaccination tests have also been made under field conditions, cfr. R. Nielsen, Nord. Vet. Med. 28 (1976), 337-348. In these tests 6 or 24 hours old agar plate cultures of H. pleuropneumoniae were used as an antigen. The cells were killed by formaldehyde and the vaccine contained 10 cells per ml. in phosphate buffered saline (physiological); (in the following for short PBS) containing 0.2% formaldehyde and with an adjuvant added. The vaccine was administered as subcutaneous injections of 2 times 4 mis. or 2 times 2 mis. with an interval of 14 days. The first vaccination was made when the pigs were 9 weeks old. 2 weeks after the second vaccination the pigs were infected with 10 H.
  • pleuropneumoniae bacteria pleuropneumoniae bacteria.
  • An object of the invention is to provide a new and improved vaccine for effectively controlling pleuropneumonia in pigs, thereby also providing a new and improved adjuvant for such a vaccine without the side effects associated with the prior art adjuvants.
  • Another object of the invention is to provide a new and improved fermentation process for the preparation of the antigen necessary for the production of the vaccine of this invention so as to make it possible to produce an effective vaccine in amounts sufficient for an extensive vaccination when desirable.
  • a further object of the invention is to provide a new and improved substrate for .the cultivation of H. pleuropneumoniae so as to obtain a growth of the microorganism which is substantially better than when cultivating on prior art substrates.
  • a still further object of the invention is to provide a method for combating pleuropneumonia in pigs by administering the vaccine of this invention.
  • a vaccine which comprises cells of Haemophilus pleuropneumoniae, parts of such cells, extracts and/or metabolism products thereof, obtained by cultivating a strain of H. pleuropneumoniae in a liquid culture based upon the substrate disclosed by S.M. Cohen and M.W. Wheeler in Am. J. Publ. Hlth. 36 (1946), 371-376, said vaccine also comprising as an adjuvant aluminium hydroxide gel and/or Freund's Incomplete or Complete Adjuvant and/or Bordetella pertussis vaccine, biomass and/or extracts thereof, as well as a sterile puffer solution as a diluent.
  • the bacterium used for the production of the vaccine of this invention is a strain of Haemophilus pleuropneumoniae which is a known microorganism deposited in various culture collections, cfr. below, and available therefrom.
  • H. pleuropneumoniae cfr. M. Kilian et al., loc. cit. (1978). This literature reference also explains why the name H. pleuropneumoniae is considered the correct name of the organism causing pleuropneumonia in pigs and gives a general survey of the characteristics of the microorganism.
  • serotype 1 in the American Type Culture Col lection, Rockville, Md., U.S.A., under No. ATCC 27088, serotype 2 under No. ATCC 27089, and serotype 3 under No. ATCC 27090.
  • serotype 2 has been deposited in the National Collection of Type Cultures, Colindale, London, U.K., under No. NCTC 10976, and. these three serotypes have all been deposited in the Czechoslovak Collection of Microorganisms, Brno, Czechoslovakia, under the Nos.
  • H. pleuropneumoniae bacteria are small Gram-negative immobile rods which may take a number of various shapes, dependent upon the culture substrate used and the environmental conditions.
  • H. pleuropneumoniae bacteria are shaped as small, coccoidal rods, and the individual colonies have a diameter of up to 3 mms. after 48 hours at 37oC. Fermented in the nutrient broth of this invention (composition, cfr. Table IV below), H. pleuropneumoniae bacteria are short rods which are often in shorter or longer chains.
  • H. pleuropneumoniae bacteria behave as described by M. Kilian, J. Gen . Microbiol. 93 (1976), 9-62, having the following characteristicproperties which together make them different from other Haemophilus species: V-factor-dependent, able to synthesize porphyrine from delta-aminolevulinic acid, produce urease and able to ferment mannitol, xylose and desoxyribose.
  • H. pleuropneumoniae has been used which as isolated from a pig having pleuropneumonia.
  • This strain was of serotype 2, cfr. R. Nielsen, loc. cit (1976), and was submitted to Statens Veterinaere Serumlaboratorium (State Veterinary Serum Laboratory), Copenhagen, Denmark, in which it was given the designation Subculture 4226.
  • the substrate used in the practice of this invention is based upon the substrate disclosed by S.M. Cohen and M.W. Wheeler, loc. cit., having the composition given in Table II below.
  • this substrate is modified so as to improve the growth of H. pleuropneumoniae and provide a substrate which is suitable for the preparation of a H. pleuropneumoniae vaccine in amounts sufficient for extensive vaccination, if desired.
  • Two different modifications are used for cultivation on agar plates and fermentation in liquid medium, respectively.
  • a substrate comprising casamino acid, yeast extract, glucose and ⁇ -nicotinamide-adenine-dinucleotide (in the following called NAD) as essential ingredients is used.
  • NAD ⁇ -nicotinamide-adenine-dinucleotide
  • this substrate should contain suitable salts and other nutrient ingredients conventionally used when cultivating microorganisms, in particular Haemophilus species.
  • An especially preferred substrate has the composition given in Table III below. Table III Substrate for agar plates
  • Glucose (Pharmacopoea Nordica) 2.0 gs.
  • Agar-agar (Difco Manual) 16.0 gs.
  • This substrate is prepared in the following manner: Casamino acid, yeast extract, glucose, NaCl, KH 2 PO 4 , and MgCl 2 ⁇ 6H 2 O are dissolved in 950 mis. ion-exchanged H 2 O. pH is adjusted to 7.2 by the addition of 1 N NaOH. CaCl 2 , FeSO 4 ⁇ 7H 2 O, cystein ⁇ HCl, and agar-agar are added and the volume is adjusted to 1000 mis. by the addition of ion--exchanged H 2 O. The solution is heated to boiling (in order to dissolve the agar), autoclaved at 121oC. and a pressure of 2 atmospheres for 15 minutes.
  • the substrate of this invention for use in the fermentation in a liquid medium also comprises casamino acid, yeast extract, glucose and NAD as essential ingredients.
  • This substrate too should contain suitable salts and other nvtrient ingredients commonly utilized in the fermentation of microorganisms, in particular Haemophilus species.
  • An especially preferred substrate has the composition given in Table IV below. Table IV
  • Glucose (Pharmaceopoea Nordica) 11.0 gs.
  • This substrate is prepared in the following manner: Casamino acid, yeast extract, glucose, NaCl, KH 2 PO 4 , and MgCl 2 ⁇ 6H 2 O are dissolved in 950 mis. ion-exchanged H 2 O. pH is adjusted to 7.2 by the addition of 1 N NaOH. CaCl 2 , FeSO 4 ⁇ 7H 2 O, CuSO 4 ⁇ 5H 2 O and cystein ⁇ HCl are added, the volume is adjusted to 1000 mis. by the addition of ion-exhanged H 2 O, and the solution is autoclaved at 121oC. and a pressure of 2 atmospheres for 15 minutes. After cooling, NAD is added and the substrate is ready for use.
  • Both of the substrates disclosed in Tables III and IV comprise assimilable sources of carbon and nitrogen which can be utilized by the microorganism H. pleuropneumoniae. On both substrates, an abundant and dense growth of the organism is obtained.
  • the fermentation procedure is as follows.
  • strains of bacteria are stored in a lyophillized state in ampoules prepared by one of the methods described below.
  • the storage is effected in a refrigerator at 5°C.
  • Method A The bacteria were cultivated at 37oC. for 18 hours on agar plates with a substrate having a composition as set out in Table III. The cells were harvested by the addition of 1 ml. PBS (composition given in Table V below) and subsequent abrasion of the cells. The cells were transferred to ampoules which were immediately cooled to -25oC. and then transferred to a lyophilizer and lyophilized.
  • PBS composition given in Table V below
  • the salts are dissolved in distilled water and autoclaved at 121oC. and a pressure of 2 atmospheres for 15 minutes. pH is 7.3.
  • Method B This method was similar to Method A, however 1 ml. skim-milk was used for each plate instead of PBS. The skim-milk had been heat-treated by heating to 100o C. for 15 minutes on 3 subsequent days .
  • Method C The bacteria were cultivated for 6 hours on a water-bath in the CAY-substrate, cfr. Table IV. The fully developed suspension of bacteria was centrifuged and the bacterial cells were resuspended in sterile skim-milk and transferred to ampoules which were cooled and lyophilized as described for Method A. The bacteria are restored from the lyophilized ampoules by the addition of substrate followed by inoculation on agar plates and in tubes containing liquid substrate. The living cultures are maintained by a daily inoculation on fresh agar plates containing the substrate of Table III. The plates are incubated in an incubator at 37°C.
  • a preculture is prepared by propagation of the bacteria. This is done by inuculation from an agar plate into a flask containing 50 mis. of the liquid CAY-substrate. The flask is incubated in an incubator at 37oC. for 8 hours. 10 mis. of medium is then withdrawn for inoculation of the second preculture.
  • the second preculture is 500 mis. of the liquid CAY-substrate in a 1 liter conical flask which is incubated on a water-bath with continuous shaking. The temperature is 32oC. and the incubation period is 18 hours.
  • the second preculture is used for the main fermentation in which the liquid CAY-substrate of Table IV is used as the fermentation medium.
  • the medium is prepared as described above for the CAY-substrate, i.e. by dissolving casamino acid, yeast extract, glucose, NaCl, KH 2 PO 4 , and MgCl 2 ⁇ 6H 2 O in ion-exchanged water, adjusting the pH to about 7.2, adding the remaining ingredients except for the NAD, autoclaving the medium at a temperature of at least about 121oC. and at a pressure of at least about 2 atmospheres for at least 15 minutes, cooling to a temperature of not more than 37oC , controlling and readjusting pH to about 7.2, and adding the NAD.
  • the fermentor is then inoculated with the second preculture of H. pleuropneumoniae obtained as described above.
  • a temperature of preferably about 37oC. is maintained and an air flow through the medium is used in order to maintain a suitable oxygen concentration in the medium. This may be done partly by controlling the air flow rate and partly by controlling the speed of the stirring device of the fermentor, thereby taking into consideration that these parameters are mutually dependent, inasmuch as an increase in any of them results in an increased oxygen concentration which in turn may necessitate a decrease in the other parameter.
  • the oxygen concentration may vary from about 1 to about 30 ppm, the most suitable concentration being from about 8 to about 20 ppm, in particular from about 8 to about 12 ppm, more specifically from about 10 to about 12 ppm.
  • the air flow rate and stirring speed necessary in order to maintain such an oxygen concentration will of course depend upon the dimensions, equipment and arrangement of the fermentor, but it has been found that an air flow rate of up to about 0.5 liter of air per minute per liter of fermentation medium, in particular from about 0.15 to about 0.4 liter, more specifically from about 0.2 to about 0.3, liter, of air per minute per liter fermentation medium is most suitable in a fermentor having a capacity of about 20 liters.
  • a suitable stirring speed is from about 200 to about 600 r.p.m., in particular about 500 r.p.m.
  • a suitable pH is in the range of from about 6.0 to about 7.9, in particular from about 6.9 to about 7.5, more specifically from about 7.1 to about 7.4, preferably at about 7.3.
  • a base suitably an aqueous sodium hydroxide solution, when necessary.
  • anti-foam agent it is not preferred to add an anti-foam agent, but it may be necessary to do so.
  • Any conventional anti-foam agent for use in fermentation processes is suitable for this purpose, in particular a silicone anti-foam agent.
  • the Kirtor is preferably provided with means which allow an automatical addition of pH-controlling base and/or anti-foam agent during the fermentation. However, the addition may also be accomplished manually, when necessary.
  • samples are withdrawn intermittently and used for a determination of the cell density.
  • the density was measured by a "Corning Colorimeter 252" at a wave length of 600 nm and expressed as number of colony forming units (CFU) by means of a reference standard curve.
  • This density measurement serves the purpose of drawing a growth curve which allows the interruption of the fermentation at the time most suitable for the subsequent production of a vaccine, i.e. when the phase in which the bacteria show an exponential growth has expired.
  • a concentrated formaldehyde solution and an Alhydrogel solution are added to the fermentation medium and after stirring for a substantial time, e.g. over night, the suspension is subjected to a centrifugation resulting in a formaldehyde-containing supernatant and a precipitate comprising H. pleuropneumoniae cells and Alhydrogel.
  • the precipitate is collected in a sterile vessel. A sample is withdrawn for controlling that the bacteria have been killed.
  • This Example shows the fermentation of Haemophilus pleuropneumoniae, strain ATCC 27089, Subculture 4226, on the CAY-substrate of this invention.
  • the fermentor was a "Biomat E 20" (Moeller &
  • Jochumsen, Vejle, Denmark having a capacity of containing 20 liters substrate in each fermentation and..being equipped with means for stirring, said means allowing a manual variation of the stirring speed, an instrument which continuously shows the stirring speed (r.p.m.), means for supplying sterile air, means for a continuous measurement and recording of the oxygen concentration of the fermentation medium, means for a continuous measurement and automatic adjustment of the temperature to a pre-determined value, means for a continuous measurement and recording of the pH of the fermentation medium and for manual or automatic addition of pH-controlling bases or acids, means for controlling the foaming in the fermentor and for the automatic addition of anti-foam agent, when necessary, and means for the withdrawal of samples during the fermentation.
  • the fermentor was charged with 17 liters ion-exchanged water and stirring was started. Then 306 gs. casamino acid, 238 gs. yeast extract, 187 gs. glucose,
  • the fermentor was inoculated with a 600 mis. second preculture of H. pleuropneumoniae prepared from a lyophilized bacteria stock and cultivated as described above.
  • the fermentation period was 7 hours and 50 minutes and samples were intermittently withdrawn and used for determint ing the absolute cell density of the fermentation medium.
  • the density was measured on a "Corning Colorimeter 252" at a wave length of 600 nm and then converted into cells per ml. by using a standard reference and a conversion table which made it possible to draw a growth curve and to determine when the exponential growth phase of the bacteria had come to an end.
  • This fermentation was carried out primarily as an experimental fermentation and therefor it was allowed to proceed in a somewhat "abnormal" manner, which involved that the pH was not adjusted during the fermentation and that no anti-foam agent was added.
  • the oxygen concentration which preferably should be 8 to 10 ppm decreased drastically during the fermentation, in particular in the exponential growth phase. This was counteracted by increasing partly the air flow rate and partly the stirring speed. It will also be seen that pH decreased to a final pH of 5.30.
  • Fig. 1 of the drawings also makes it possible to calculate the time (Tg) necessary for doubling the contents of bacteria in the fermentation medium during the exponential growth phase.
  • Tg is given by the formula
  • C 2 is the number of cells per unit volume at the time T 2 after inoculation
  • C 1 is the number of cells per unit volume at the time T 1 after inoculation
  • Example 2 This example shows a "normal" fermentation which involves that pH is continuously and automatically adjusted to about 7.2, that anti-foam agent is automatically added when necessary, that a more vigorous stirring is maintained, and that the oxygen concentration is continuously held at a relatively high level.
  • the fermentor was identical to the fermentor used in Example 1.
  • the volume of the fermentation medium was about 20 liters of which the inoculation medium provided 0.5 liter, the balance being 19 liters ion-exchanged water containing casamino acid, yeast extract, glucose and salts in amounts providing a CAY-substrate of the relative composition given in Table IV.
  • the charging of the fermentor, the autoclaving, cooling and subsequent addition of NAD was carried out as described in Example 1.
  • the initial pH of the medium was 7.1 and the temperature at the inoculation was 31oC.
  • the inoculation medium was 500 mis. of a second preculture of H. pleuropneumoniae, strain ATCC 27089, Subculture 4226, cultivated in the manner described above.
  • the pH was maintained at an almost constant value of about 7.2 by the automatic addition of a 1 N NaOH solution when necessary.
  • Stirring was performed at 500 r.p.m. and the oxygen concentration was maintained at from about 8 to about 12 ppm. This was achieved by increasing the air flow rate when necessary.
  • An anti-foam agent having the trade name "Silicone RS Antifoam C. DAK" was automatically added when necessary.
  • Example 1 results in the lowest initial Tg, which means a faster growth of the bacteria in the fer mentor and consequently a shorter fermentation period before the broth is ready for utilization for the production of a
  • H. pleuropneumoniae vaccine also results in a faster transition into the stationary growth phase, viz. after a fermentation period of about 5 hours, whereas the "normal" fermentation results in a prolonged period of exponential growth phase of the bacteria, viz. more than 7 1/2 hours.
  • Example 2 37 ⁇ 10 8 cells per ml. compared to about 27 ⁇ 10 8 cells per ml. in Example 1 (sample No. 18 where the exponential growth phase ends). Therefore, from a commercial point of view (vaccine production) the "normal" fermentation process in Example 2 is preferred because the greater time consumption is more than counterbalanced by the higher yield of cells utilizable in the subsequent vaccine production.
  • This Example is a Comparison Example which demonstrates that fermentation of H. pleuropneumoniae, Subculture 4226, on a known substrate results in a much slower growth rate and a much lower yield than fermentation in the substrate of this invention.
  • the fermentor was identical to the fermentor used in Examples 1 and 2.
  • the substrate was a commercial "CASO-bouillon Merck” which is described as No. 5478 in "Handbook of Microbiology” published by E. Merck. Darmstadt, Federal Republic of Fermany, to which 6 mgs. NAD per liter bouillon had been added.
  • the fermentor was charged with 17 liters of
  • the fermentation period was 6 hours 35 minutes and during this period samples were withdrawn and analyzed in the. manner and for the parameter described in Example 1.
  • the results of the fermentation are summarized in Table VIII.
  • Example 2 After a fermentation period of 7 1/2 hours, the fermentation described in Example 2 was stopped by the addition of 60 mis. concentrated formaldehyde solutionand 600 mis. of Alhydrogel.
  • the Alhydrogel added was a
  • the precipitate collected as described in Example 4 was used for the production of a H. pleuropneumoniae vaccine.
  • a vaccine was obtained by diluting the precipitate with phosphate buffered saline (physiological) (PBS) solution of the composition shown in Table V to about the desired density of the vaccine. Then "Thiomersal” [sodium 2-(ethylmercurithio)benzoate] and optionally Alhydrogel were added and the precipitate, PBS solution, "Thiomersal” and Alhydrogel were suspended in a sodium chloride-phosphate buffer having a pH of 7, stirred for 20 minutes and a sample withdrawn for analysis. On the basis of this analysis, the density of the vaccine was adjusted to the desired value. Then a Bordetella pertussis suspension (a B. pertussis vaccine) was added and stirring continued for 30 minutes. The density was controlled and the vaccine was filled into capped vials.
  • PBS phosphate buffered saline
  • a suitable density of the vaccine is about 20 I.O.U. and consequently it is preferred that the precipitate of Example 4 is diluted with PBS solution to about this value and that the final density of the vaccine is adjusted to a level of 20 I.O.U. before the addition of the B. pertussis suspension. It has also been found that a suitable concennration of the adjuvant "Thiomersal" is about 0.01 % w/v and that the adjuvant Alhydrogel should preferably be present in a concentration of about 3 % w/v.
  • the B. pertussis vaccine per se biologicalmass and/or extracts
  • the method for its production form part of the present invention which involves that any such vaccine is utilizable as adjuvant in the H. pleuropneumoniae vaccine of the invention
  • the B. pertussis vaccine as well as the final vaccine of this invention meet all requirements set out for vaccines according to national and/or international standards.
  • the preferred B. pertussis vaccine for use in the production of a H. pleuropneumoniae vaccine of this invention is a Vaccinum pertussis which has been prepared, identified and tested as described in the European Pharmacopoeia,
  • a suitable amount of the adjuvant B. pertussis suspension in the H. pleuropneumoniae vaccine of the invention is about 16 I.O.U. vaccine which may be conveniently obtained by the additon of 100 mis.
  • B. pertussis suspension having a density of 160 I.O.U. to 1 liter of suspension obtained by mixing the precipitate of Example 4, PBS solution, "Thiomersal", Alhydrogel when used, and sodium chloride-phosphate buffer.
  • Vaccinum pertussis Ph. Eur. 160 I.O.U. 0.1 ml.
  • the vaccine of this invention has been found to be effective in combating pleuropneumonia in pigs exposed to infection by the microorganism Haemophilus pleuropneumoniae and it is thus indicated for the prophylactic treatment of pigs, in particular seronegative pigs, before they are moved to an environment which may already be infected by H. pleuropneumoniae or where a risk of such an infection may exist.
  • the pigs are vaccinated with a H. pleuropneumoniae vaccine of this invention by intramuscular or subcutaneous infection of the vaccine, subcutaneous injection being preferable.
  • the vaccine of this invention is administered twice with a suitable period between the injections, preferably not less than 3 weeks.
  • the dosage administered will of course depend upon the body weight of the pigs, the potency of the vaccine, the risks of side effects and the nature of such effects when present, and other factors commonly taken into consideration when effecting a vaccination, irrespective of the active substance in the vaccine.
  • a dosage of 2 mis. of the vaccine described in Example 6 has been found to be effective in general in pigs having a body weight of up to 30 kgs.
  • a dosage of 4 mis. of the vaccine is suitable.
  • the following Examples show the advantages obtained by using the vaccine of this invention for vaccination against pleuropneumonia compared to prior art vaccines comprising other adjuvants.
  • mice 45 pigs weighing about 25 kgs. were given a subcutaneous injection of the vaccines A to E described in more detail below. 3 weeks later the vaccination was repeated. The dosage was 2 mis., 5 mis., and 6 mis., respectively. 3 weeks after the second injection the pigs were challenged with H. pleuropneumoniae bacteria (approximately 10 H. pleuropneumoniae bacteria per animal) and 3 pigs which had received no vaccination were also challenged and used as controls. The control animals died, and the vaccinated animals survived. After slaughtering, the animals were examined in order to establish whether the infection had resulted in changes of the lungs and/or changes which, might affect the value of the slaughtered animal as being fit for human consumption.
  • H. pleuropneumoniae bacteria approximately 10 H. pleuropneumoniae bacteria per animal
  • the vaccines used in this test vaccination were the following:
  • A. H. pleuropneumoniae cells were cultivated on agar plates and harvested by a PBS solution (composition, cfr. Table V) containing a 0.01 % w/v solution of "Thiomersal". The cell suspension containing about 10 10 cells per ml. was extracted for 30 minutes at 56oC. and then centrifuged and 1 part by volume of Freund' s Incomplete Adjuvant was added to 3 parts by volume of supernatant. Dosage 2 mis. (twice). B. As vaccine A except that the extracted cells were boiled at 100°C. for 2 hours before centrifugation. Dosage 2 mis. (twice)
  • the cells were cultivated as vaccine A, harvested by PBS solution containing a 0.01 % w/v solution of "Thiomersal" and adjusted to a density of 20 I.O.U. and then 50 % of Freund's Incomplete
  • Adjuvant was added. Dosage 5 mis. (twice) containing about 10 9 H. pleuropneumoniae cells per ml.
  • D. H. pleuropneumoniae cells were cultivated in a flask until the density was 5 U.O.U., killed by formaldehyde and precipitated by the addition of
  • vaccine E of this invention is superior to the prior art vaccines A to D in providing protection against infections caused by
  • Vaccine E is the only one in which neither symptoms nor changes of lungs and/or remarks by slaughtering are observed. Changes of the lungs, viz. scars in the lung tissue as a result of a pleuropneumonia attack, were observed in 8 of the 45 vaccinated animals. By slaughtering chronical pleurisy was remarked in 4 of the animals. However, all of the 45 animals were acceptable for human consumption.
  • a further test vaccination was carried out on 46 pigs and 2 animals were used for control purposes. The animals weighed about 25 kgs. The vaccinations and challenge infections were carried out in the manner described in Example 7. The dosages used for vaccination were 2 times 2 mis.
  • the primary purpose of this test is to compare the effect of the Bordetella pertussis adjuvant of this invention to the effect of various other adjuvants of which some have already been used as such adjuvants in the prior art.
  • compositions of the test vaccines are given below. It should be noted that "vaccine” L is no vaccine in the common sense of this term, however, the results obtained demonstrate that pleuropneumonia was not successfully controlled by pernasal and peroral administration of the active ingredient in the vaccine of this invention.
  • the vaccines used were the following:
  • Example 7 A vaccine of the composition of Example 6 (vaccine E) , except for a content of "Quil A” as the adjuvant instead of Vaccinum pertussis.
  • Quil A is a saponine derivative of known composition which has been disclosed by K. Dalsgaard in Acta Vet. Scand., Suppl. 69 (1978).
  • Bortavac is a commercial Bordetella bronchiseptica vaccine sold by the producer, Kitasato, Tokyo, Japan.
  • Levoripercol is Levamisolum NFN anthelminticum sold by the firm Lundbeck, Copenhagen, Denmark. Levamisolum is a Non-Proprietary Name approved by NFN (The Nordic Pharmacopoeia Council).
  • LPF Proliferative Factor
  • L. H. pleuropneumoniae cells were cultivated on an agar plate, harvested and transferred into an aerosol which were atomized into the nose and mouth of the animals.
  • vaccines E, I and K are within the scope of the present invention.
  • Table X shows that vaccination with a H . pleuropneumoniae vaccine comprising killed H . pleuropneumoniae bacteria prepared along the lines disclosed in Examples 2, 4, and 5 above as the active ingredient is very effective for protecting pigs against pleuropneumonia attack.
  • a H . pleuropneumoniae vaccine comprising killed H . pleuropneumoniae bacteria prepared along the lines disclosed in Examples 2, 4, and 5 above as the active ingredient is very effective for protecting pigs against pleuropneumonia attack.
  • pertussis vaccine adjuvant or biomass and/or extracts thereof used according to the invention is superior and thus preferred because it results in an optimal protection and no or only a slight and short reaction on the injection site whereas the prior art adjuvants tend to give rise to granuloms on the injection site.

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Abstract

Dans un procede ameliore de lutte contre la pleuropneumonie du cochon par inoculation d'un vaccin comprenant des cellules du microorganisme connu et generalement disponible Hemophilus pleuropneumoniae, des parties de telles cellules, des extraits et/ou des produits du metabolisme de ceux-ci en tant que substances actives, des adjuvants et un tampon, l'amelioration provenant de l'utilisation d'un vaccin de Bordetella pertussis, d'une biomasse et/ou d'extraits de celui-ci en tant qu'adjuvant, en meme temps que le nouveau vaccin a H. pleuropneumoniae comprenant un adjuvant base sur du B, pertussis. Le procede et le vaccin entrainent une protection amelioree des cochons contre des attaques de pleuropneumonie sans effets secondaires. De plus est decrit un substrat nouveau et ameliore denomme substrat "CAY", pour la culture de microorganismes, en particulier la bacterie H. Pleuropneumoniae, lequel en comparaison des substrats connus, resulte en un rendement sensiblement plus eleve en biomasse appropriee pour la production de vaccin de H. pleuropneumoniae, ce substrat comprenant de l'acide casaminique, de l'extrait de levure, du glucose et du dinucleotide-nicotinamide-adenine (NAD) en tant que substances essentielles et il se prete a la culture de H. pleuropneumoniae en milieu solide aussi bien qu'en milieu liquide par de petites modifications dans la composition du substrat. Un procede ameliore pour la fermentation aerobie de H. pleuropneumoniae en milieu liquide comprenant le substrat "CAY", cette fermentation etant avantageusement effectuee a une temperature d'environ 37 C, un pH d'environ 7,1 a 7,4 et a une concentration en oxygene dans le milieu de fermentation d'environ 8 a 12 ppm, et en maintenant la concentration desiree en oxygene par variation de la vitesse d'agitation et/ou du debit d'air.
PCT/DK1980/000021 1979-04-04 1980-04-02 Vaccin pour lutter contre la pleuropneumonie chez le cochon procede et substrat pour la fermentation aerobie hemophilus pleuropneumoniae WO1980002113A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
DE19803041448 DE3041448A1 (de) 1979-04-04 1980-04-02 A vaccine for combatting pleuropneumonia in pigs,and a process and a substrate for the aerobic fermentation of haemophilus pleuropneumoniae

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DK138379A DK138379A (da) 1979-04-04 1979-04-04 Vaccine imod pleuropneumonia (ondartet lungesyge) hos svin dens anvendelse samt fremgangsmaade og substat til dyrkning specielt aerob fermentering af mikroorganismen haemophilus pleuropneumoniae
DK1383/79 1979-04-04

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WO1980002113A1 true WO1980002113A1 (fr) 1980-10-16

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Country Status (10)

Country Link
EP (1) EP0026209A1 (fr)
JP (1) JPS56500339A (fr)
AT (1) ATA903480A (fr)
BE (1) BE882619A (fr)
DK (1) DK138379A (fr)
GB (1) GB2057882B (fr)
IE (1) IE49754B1 (fr)
NL (1) NL8020132A (fr)
SE (1) SE8008500L (fr)
WO (1) WO1980002113A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987007148A1 (fr) * 1986-05-23 1987-12-03 Midcon Labs, Inc. Co-vaccination utilisant une preparation de bacteries gram-negatives sans chaines laterales o-hydrate de carbone
EP0354628A1 (fr) * 1988-08-12 1990-02-14 Centraal Diergeneeskundig Instituut Vaccin pour la prévention, respectivement le contrôle de la maladie provoquée chez les cochons par heamophilus pleuropneumoniae ainsi qu'une méthode pour sa production
FR2652266A1 (fr) * 1989-09-26 1991-03-29 Rhone Merieux Vaccin protecteur contre l'hemophilose porcine.
EP0453024A1 (fr) * 1990-04-20 1991-10-23 Akzo Nobel N.V. Vaccin de sous-unités d'actinobacillus pleuropneumoniae
US5925354A (en) * 1995-11-30 1999-07-20 Michigan State University Riboflavin mutants as vaccines against Actinobacillus pleuropneumoniae
WO2010042970A1 (fr) * 2008-10-15 2010-04-22 Pork Crc Ltd Procédé de vaccination de porcs

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2725204A1 (de) * 1976-06-04 1977-12-22 Merieux Inst Immunitaets-stimulierendes medikament und verfahren zu seiner herstellung
DE2825464A1 (de) * 1977-06-10 1978-12-21 Kakenyaku Kako Kk Biologisch aktive substanz, verfahren zu deren herstellung und dieselbe enthaltendes pharmazeutisches mittel

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2725204A1 (de) * 1976-06-04 1977-12-22 Merieux Inst Immunitaets-stimulierendes medikament und verfahren zu seiner herstellung
DE2825464A1 (de) * 1977-06-10 1978-12-21 Kakenyaku Kako Kk Biologisch aktive substanz, verfahren zu deren herstellung und dieselbe enthaltendes pharmazeutisches mittel

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
American Journal of Public Health and the Nations Health, Volume 36, issued 1946, pages 371-376. *
International Journal of Systematic Bacteriology, Volume 28, issued 1978, pages 20-26. *
Nordisk Veterinarmedicin, Volume 28, issued 1976, pages 337-348. *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987007148A1 (fr) * 1986-05-23 1987-12-03 Midcon Labs, Inc. Co-vaccination utilisant une preparation de bacteries gram-negatives sans chaines laterales o-hydrate de carbone
EP0354628A1 (fr) * 1988-08-12 1990-02-14 Centraal Diergeneeskundig Instituut Vaccin pour la prévention, respectivement le contrôle de la maladie provoquée chez les cochons par heamophilus pleuropneumoniae ainsi qu'une méthode pour sa production
US5254340A (en) * 1988-08-12 1993-10-19 Centraal Diergeneeskundig Instituut Vaccine suitable for prophylaxis and control, respectively, of the pig disease caused by Haemophilus pleuropneumoniae and a method for obtaining extracellular proteinaceous material of Haemophilus pleuropneumoniae for use in such vaccines
FR2652266A1 (fr) * 1989-09-26 1991-03-29 Rhone Merieux Vaccin protecteur contre l'hemophilose porcine.
EP0420743A1 (fr) * 1989-09-26 1991-04-03 Rhone Merieux Vaccin protecteur contre l'hémophilose porcine
WO1991004747A1 (fr) * 1989-09-26 1991-04-18 Rhone Merieux Vaccin protecteur contre l'hemophilose porcine
AU635349B2 (en) * 1989-09-26 1993-03-18 Merial Protection vaccine against swine hemophilosis
EP0453024A1 (fr) * 1990-04-20 1991-10-23 Akzo Nobel N.V. Vaccin de sous-unités d'actinobacillus pleuropneumoniae
US5648081A (en) * 1990-04-20 1997-07-15 Akzo Nobel N.V. Actinobacillus pleuropneumoniae subunit vaccine
CN1055024C (zh) * 1990-04-20 2000-08-02 阿克佐公司 胸膜肺炎放线杆菌亚单元疫苗
US5925354A (en) * 1995-11-30 1999-07-20 Michigan State University Riboflavin mutants as vaccines against Actinobacillus pleuropneumoniae
WO2010042970A1 (fr) * 2008-10-15 2010-04-22 Pork Crc Ltd Procédé de vaccination de porcs

Also Published As

Publication number Publication date
EP0026209A1 (fr) 1981-04-08
IE800696L (en) 1980-10-04
GB2057882A (en) 1981-04-08
IE49754B1 (en) 1985-12-11
SE8008500L (sv) 1980-12-03
BE882619A (fr) 1980-10-03
ATA903480A (de) 1984-05-15
JPS56500339A (fr) 1981-03-19
GB2057882B (en) 1983-05-18
DK138379A (da) 1980-10-05
NL8020132A (nl) 1981-01-30

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