EP0026209A1 - A vaccine for combatting pleuropneumonia in pigs, and a process and a substrate for the aerobic fermentation of haemophilus pleuropneumoniae - Google Patents

Abstract

In an improved method of controlling pig pleuropneumonia by inoculating a vaccine comprising cells of the known and generally available microorganism Hemophilus pleuropneumoniae, parts of such cells, extracts and / or metabolic products thereof as as active substances, adjuvants and a buffer, the improvement resulting from the use of a Bordetella pertussis vaccine, a biomass and / or extracts thereof as an adjuvant, together with the new H. pleuropneumoniae vaccine comprising an adjuvant based on B, pertussis. The method and the vaccine provide improved protection of pigs against attacks of pleuropneumonia without side effects. In addition, a new and improved substrate, called the "CAY" substrate, is described for the culture of microorganisms, in particular the H. Pleuropneumoniae bacteria, which, in comparison with known substrates, results in a significantly higher yield of biomass suitable for the production of H. pleuropneumoniae vaccine, this substrate comprising casaminic acid, yeast extract, glucose and dinucleotide-nicotinamide-adenine (NAD) as essential substances and it is suitable for the cultivation of H. pleuropneumoniae in solid as well as in liquid medium by small modifications in the composition of the substrate. An improved process for the aerobic fermentation of H. pleuropneumoniae in a liquid medium comprising the substrate "CAY", this fermentation being advantageously carried out at a temperature of approximately 37 C, a pH of approximately 7.1 to 7.4 and at a oxygen concentration in the fermentation medium of about 8 to 12 ppm, and maintaining the desired oxygen concentration by varying the stirring speed and / or the air flow.

Description

A VACCINE FOR COMBATING PLEUROPNEUMONIA IN PIGS, AND A PROCESS AND A SUBSTRATE FOR THE AEROBIC FERMENTATION OF HAEMOPHILUS PLEUROPNEUMONIAE.

Background of the Invention In the early 1960's 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. As adjuvants 15 % Alhydrogel (aluminium hydroxide gel) and Freund's Incomplete Adjuvant, cfr. J. Freund, Am. Rev. Microbiol. 1 (1947), 291, respectively were used.

The results of these vaccination tests are summa rized in the following Table I .

Table I

Age of antigen Adjuvant Protection (%)

6 hours 15 % Alhydrogel 67

Freund's Incompl. 90

24 hours 15 % Alhydrogel 25

Freund's Incompl. 67

It should be added that the use of Freund's Incomplete Adjuvant gave rise to a formation of granulomes at the injection site.

Attempts have also been made in order to obtain a protection by using 20 hours cultures killed by formaline as the antigen and Alhydrogel and Freund's Complete Adjuvant, respectively, as the adjuvant. The dosis injected was 2 times 5 mis. containing 10⁹ bacteria per ml. The first injection was made intramuscularly on 50 days old pigs and the second injection 1 week later. 1 week thereafter, the pigs were infected. The result was a significant evidence that the vaccine had a protecting effect, but no significant evidence that one adjuvant had advantages above the other.

E. Scholl et al., cfr. Proc. Int. Pig Vet. Soc. Congress, Ames, Iowa, U.S.A. (1976), have used heat killed cultures of H. pleuropneumoniae as antigens and have vaccinated by two methods: 1) by .increasing doses, viz. 0.5, 1 and 3 mis., and 2) by 2 times 5 mis. All injections were made with an interval of 2 weeks. Serological test of blood samples showed that repeated injections of small doses resuited in antibodies in the blood, but no sensitized lymphocytes, whereas injections of high doses resulted in sensitized lymphocytes, but no antibodies in the blood.

The same workers, cfr. Proc. Int. Pig Vet. Soc. Congress, Zagreb, Yugoslavia (1978), have used cultures of H. pleuropneumoniae killed by formaline in concentrations of 10⁹ bacteria per ml. The vaccination doses and the method of vaccination were the same as in the earlier reported work, but in this later work a group of placebo pigs was included and the animals were put into a chronically infected herd of pigs. Clinical symptoms were observed and following the slaughtering the lungs and the thoracic cavity were examined. The results showed a good effect of the vaccination, but no differences between the two methods of vaccination.

M.F. de Jong, cfr. Proc. Int. Pig Vet. Soc. Congress, Zagreb, Yugoslavia (1978), has made vaccination tests with 24 hours H . pleuropneumoniae cultures to which an incomplete adjuvant on the basis of mineral oil had been added. For each dosis 20 mgs. (wet weight) antigen were used and two injections were made with an interval of 3 weeks, the first vaccination when the pigs were 10 weeks old. 3 weeks after the second vaccination, the pigs were challenged with 16 or 24 hours cultures. With 24 hours challenge cultures, the pigs became ill, but recovered within 1 week. On the other hand, the pigs challenged with a 16 hours challenge culture died within 36 hours. Challenge involves, as will be known by those skilled in the art, an experimental infection of the test animals with live cells of the infecting microorganism.

M.F. de Jong, loc. cit., then used 6 hours cultures for both vaccination and challenge. The pigs were challenged with different amounts of cells and the mortality rate was dependent upon the number of cells used. Besides, pigs having been vaccinated with 6 hours cultures were challenged with 16 hours cultures and 50 % of the pigs survived. 1 week later, the surviving pigs were infected with a 6 hours culture and now they did not show even symptoms of illness.

The works on test vaccinations mentioned above clearly show that it is necessary .to add an adjuvant to a vaccine comprising H. pleuropneumoniae antigens in order to obtain a satisfactory effect of the vaccination. The substances most commonly used as adjuvants are Alhydrogel and Freund's Adjuvant (Complete or Incomplete). Of these substances Freund's Adjuvant has until now been the most effective, but as also reported, cfr. R. Nielsen, loc. cit. (1976), it results in the formation of granulomes at the injection site, and therefor it is considered unsuitable. Thus, the problem of providing an adjuvant which is effective, but does not interfere with the animals has not been solved by the previously reported test vaccinations.

Another unsolved problem is the preparation of an antigen for the production of the vaccine. This problem resides in the fact that H. pleuropneumoniae has a rather poor growth on the previously used and classical substrates which sets strongly restricted limits for the amount of vaccine which it has been possible to produce.

Thus, J. Nicolet, cfr. Zbl. Bakt. I Abt. Orig. 216 (1971), 487-495, in his serological studies has used a substrate comprising PPLO-agar (cfr. DIFCO-Manual IX, 1953, Catalogue No. 412 - PPLO = pleuropneumoniae-like organisms) to which had been added 0.1 % glucose, 2.5 % yeast extract, 5 % horse serum and 5 mgs./liter NAD (β-nicotinamide-adenine-dinucleotide). The same substate was used by R. Nielsen, loc. cit. (1976).

M.F. de Jong, loc. cit., has used Brain Heart Infusion containing NAD as the substrate, and M. Kilian et'al., cfr. Int. J. Syst. Bacteriol. 28 (1978), 20-26, have made taxonomical studies of H. pleuropneurαoniae, thereby cultivating the strains on a substrate comprising:

1 % of Neutralized Bacteriological Peptone ("Oxoid") 1 % of Yeast Extract 1 % of Glucose, 10 mgs./liter of NAD, and a salt solution as disclosed by L.V. Holdeman and

W.E.C. Moore in Anaerobe Laboratory Manual, 3rd ed., Virginia Polytechnic Institute, Anaerobe Laboratory, Blacksburg (1975).

A few other workers have cultivated H. pleuropneumoniae in liquid cultures, but with rather poor results. Summary of the Invention

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.

These and other objects are obtained by providing .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. Detailed Description of the Invention 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.

Investigations have shown that there exist at least 6 different serotypes of 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.

Of the various serotypes of H. pleuropneumoniae described in the above literature reference, some have been deposited, thus 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. Besides, 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. CCM 5869, CCM 5870, and CCM 5871, respectively. Moreover , M. Kilian et al ., loc. cit. (1978), describe serotypes designated as serotypes 4, 5, and NT. However, no deposition numbers are given.

Applicant's own investigations have confirmed that 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.

Thus, on agar plates they are most often small coccoidal or short rods whereas in liquid cultures they may vary from small coccoidal rods to longer slender or thick rods, as an exception (which is rather seldom) even to pleomorphical filament shapes. Cultured on agar plates on the substrate of this invention (composition, cfr. Table III below), 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.

In biochemical respect, 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.

More specifically, a strain of 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.

Table II

Casamino aci^{d *)} technical 10.0 gs.

NaCl 2.5 gs.

KH₂PO₄ 0.5 g.

Mgd₂ · 6H₂O 0.4 g.

Soluble starch 1.5 gs.

CaCl₂ (1 % solution) 1.0 ml.

FeSO₄·7H₂O (0.5 % solution) 2.0 mis.

CuSO₄·5H₂O (0.05 % solution) 1.0 ml.

Cystein·HCl (1 % solution) 2.5 mis.

Yeast dialysate 50.0 mis.

Distilled water to 1 liter pH adjusted to 7.2-7.3

^*) Difco Manual 1953, No. 231, "Bacto"

For the purposes of this invention, 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. For agar plates, a substrate comprising casamino acid, yeast extract, glucose and β-nicotinamide-adenine-dinucleotide (in the following called NAD) as essential ingredients is used. Besides, 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

Casamino acid , technical (Difco Manual) 6.0 gs. Yeast extract, technical (Difco Manual) 25.0 gs.

Glucose (Pharmacopoea Nordica) 2.0 gs.

NaCl 2.5 gs.

KH₂PO₄ 0.5 g.

MgCl₂ · 6H₂O 0.1 g. CaCl₂ (1 % w/v solution) 1.0 ml.

FeSO ·7H₂O (0.5 w/v solution, freshly prepared) 0.5 ml.

Cystein·HCl (1 % w/v solution) 0.8 ml.

Agar-agar (Difco Manual) 16.0 gs.

NAD (2.5 % w/v solution) 1.0 ml. (Sigma Chemical Co., St. Louis, Mo., U.S.A. )

Water to 1000 mis.

This substrate is prepared in the following manner: Casamino acid, yeast extract, glucose, NaCl, KH₂PO₄, and MgCl₂·6H₂O are dissolved in 950 mis. ion-exchanged H₂O. pH is adjusted to 7.2 by the addition of 1 N NaOH. CaCl₂, FeSO₄·7H₂O, cystein·HCl, and agar-agar are added and the volume is adjusted to 1000 mis. by the addition of ion--exchanged H₂O. The solution is heated to boiling (in order to dissolve the agar), autoclaved at 121ºC. and a pressure of 2 atmospheres for 15 minutes. After cooling to 50ºC., the NAD is added and the substrate is poured into Petri dishes. 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

Substrate for fermentation in liquid medium

Casamino acid, technical (Difco Manual) 18.0 gs.

Yeast extract, technical (Difco Manual) 14.0 gs.

Glucose (Pharmaceopoea Nordica) 11.0 gs.

NaCl 2.5 gs.

KH₂PO₄ 0.5 g MgCl₂·6H₂O 0.1 g

CaCl₂ (1 % w/v solution) 1.0 ml.

FeSO₄·7H₂O (0.5 % w/v solution, freshly prepared) 2.0 mis.

CuSO₄·5H₂O (0.05 % w/v solution) 1.0 ml.

Cystein·HCl (1 % w/v solution) 2.5 mis. NAD (2.5 % w/v solution) (Sigma Chemical Co.) 1.0 ml.

Water to 1000 mis.

This substrate, called CAY-substrate for short, is prepared in the following manner: Casamino acid, yeast extract, glucose, NaCl, KH₂PO₄, and MgCl₂·6H₂O are dissolved in 950 mis. ion-exchanged H₂O. pH is adjusted to 7.2 by the addition of 1 N NaOH. CaCl₂, FeSO₄·7H₂O, CuSO₄·5H₂O and cystein·HCl are added, the volume is adjusted to 1000 mis. by the addition of ion-exhanged H₂O, 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.

The 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.

Table V

Composition of PBS NaCl 8.0 gs.

KCl 0.2 g.

Na₂HPO₄ 1.15 gs. KH₂PO₄ 0.2 g.

Distilled water to 1 liter.

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, KH₂PO₄, and MgCl₂·6H₂O 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. During the fermentation, 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.

For the fermentation of H. pleuropneumoniae, 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. For this fermentor, a suitable stirring speed is from about 200 to about 600 r.p.m., in particular about 500 r.p.m.

It should be emphasized that these parameters are not critical in the practice of this invention, and for any fermentor those skilled in the art will be able to adjust the parameters so as to maintain the desired oxygen concentration.

During the fermentation it is preferable to maintain a substantially constant pH. 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. This may be accomplished by the addition of a base, suitably an aqueous sodium hydroxide solution, when necessary.

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 termentor 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.

During the fermentation, 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.

When the fermentation has been completed, 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.

The following Examples further illustrate the advantages obtained by cultivating H. pleuropneumoniae on the CAY-substrate of the present invention.

Example 1

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,

42.5 gs. NaCl, 8.5 gs. KH₂PO₄, and 1.7 gs. MgCl₂· 6H₂O were added. pH was adjusted to 7. 2 by the addition of a 1 N NaOH solution and 17.0 mis of a 1 % w/v solution of CaCl₂, 34.0 mis. of a 0.5 % w/v solution of FeSO₄·5H₂O (freshly prepared), 17.0 mis. of a 0.05 % w/v solution of CuSO₄·5H₂O, and 42.5 mis. of a 1 % w/v solution of cystein·HCl were added. The fermentor was closed, autoclaved for 15 minutes at 121ºC. and a pressure of 2 atmospheres and then cooled to 36°C. Then 17.0 mis. of 2.5 % w/v solution of NAD were added.

This procedure resulted in a fermentation medium having the composition given in Table IV and with a pH of 7.45.

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 results obtained from analyzing the samples are given in Table VI below.

It will be seen that 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.

The results in Table VI have been used for drawing the growth curve shown in Fig. 1 of the drawings. In the fiture, the abscissa shows the time after inoculation, and the ordinate shows the number of cells per ml. fermentation medium. It should be noted that the ordinate is depicted on a logarithmic scale. The figure shows that the exponential growth phase ends in about 5 hours after the inoculation which means that the fermentation should have been stopped at this point if the medium had been intended for use in the production of a vaccine. However, as this fermentation was an experimental fermentation, it was continued into the stationary growth phase.

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

wherein p. is represented by the formula

wherein C₂ is the number of cells per unit volume at the time T₂ after inoculation, and C₁ is the number of cells per unit volume at the time T₁ after inoculation.

In Fig. 1 the exponential growth phase is represented by the straight line part of the curve. Thus, it will be seen that this phase extends from not later than 1 hour to about 5 hours after inoculation. Using this part of the curve (the results marked with a circle in Fig. 1, i.e. samples Nos. 6 to 18) for a calculation according to formula (II) shows that

G.VPI wherein T₂ and T₁ have been expressed as hours and the common factor 10 8 has been ignored in the calculation of loge C₂ and loge C₁. According to formula (I) this means that

which equals 39.6 minutes.

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.

When the inoculation, medium had been added, the fermentation was started and continued for 7 hours 30 minutes. The inoculation medium was 500 mis. of a second preculture of H. pleuropneumoniae, strain ATCC 27089, Subculture 4226, cultivated in the manner described above. During the fermentation 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.

Samples were withdrawn during the fermentation and analyzed as described in Example 1 in order to follow the growth rate. The fermentation period was 7 hours 30 minutes.

The results of the fermentation have been summarized in Table VII.

- _* Table VII

Time Oxygen after concenStirring Temper¬

Sample inocutration speed ature Air flow rate Absolute Cells No. lation (ppm) pH (r.p.m.) (°C) (liters/min.) density per ml. Remarks

1 0^h 8 7.1 500 31 3 0.101 0 Before inoculation

2 0^h5^m 10.8 7.3 500 32 3 0.108 0.20×10⁸ After inoculation

3 0^h18^m 11.6 7.3 500 36 4 0.108 0.20

4 0^h33^m 11.6 7.3 500 37 4 0.111 0.25

5 0^h48^m 11.6 7.3 500 37 4 0.115 0.35

6 1^h20^m 11.6 7.3 500 37 4 0.118 0,45

7 1^h45^m 11.6 7.3 500 37 4 0.135 0.90

8 2^h 11.6 7.3 500 37 4 0.141 0.10 9 2^h15^m 11.6 7.25 500 37 4 0.155 ι . 45

10 2^h30^m 11.6 7.25 500 37 4 0.161 1.65

11 2^h48^m 11.6 7.25 500 37 4 0.180 2.15

12 3^h 11.6 7.25 500 37 4 0.198 2.65

13 3^h15^m 11.6 7.3 500 37 4 0.220 3.30

14 3^h45^m 11.6 7.2 500 37 4 0.242 4.00

15 4^h 11.2 7.2 500 37 4 0.290 5.50

16 4^h15^m 10.8 7.2 500 37 4 0.315 6.30

17 4^h30^m 10.8 7.15 500 37 4 0.353 7.50

18 4^h45^m 11.2 7.1 500 37 4 0.380 8.35×10⁸

The data given in Table VII have been used for drawing the growth curve shown in Fig. 2 of the drawings. The abscissa and the ordinate have the meanings given for Fig. 1, cfr. Example 1. From Fig. 2, it will be seen that the fermentation is stopped while the microorganism is still in the exponential growth phase although the growth rate is somewhat less pronounced than in the initial phase of the fermentation, a slight decrease in the slope of the curve occurring at about 3 hours 30 minutes after the inoculation.

The measurements shown with circles in Fig. 2 have been used for calculations according to the formulae (I) and (II) of Example 1, partly for the initial phase (phase 1, samples Nos . 5 to 13), and partly for the last period (phase 2, samples Nos. 15 to 27) of the fermentation.

The units used in the calculations in Example 1 were used in these calculations, too, which gave the following resuits.

which equals 45.5 minutes.

which equals 75.7 minutes.

A comparison of the results obtained according to Examples 1 and 2 will show that although the "abnormal" fermentation process of 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, it 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.

More important, the "normal" fermentation also results in a higher yield of cells per unit volume at the time where the broth is ready for vaccine production, viz. more than

37 × 10⁸ cells per ml. compared to about 27 × 10⁸ 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.

Example 3

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

CASO-bouillon, autoclaved and cooled to 37 C. as described in Example 1, and then NAD was added. The initial pH was 7.3.

As inoculation medium, 600 mis. of a second preculture of H. pleuropneumoniae ATCC 27089, Subculture 4226, was added. This preculture had been cultivated in the manner described above. During the fermentation, a temperature of 37ºC was maintained. pH was controlled manually and allowed to decrease to a final pH of 6.85. The initial oxygen concentration was 10 ppm, but it dropped very fast to 0 , partly because the air flow rate is low and partly because the stirring was stopped immediately after the inoculation and not resumed until 1 hour after an oxygen concentration of 0 had been reached. Thus, it is characteristic for this fermentation that it must take place under almost anaerobic conditions in order to obtain an acceptable yield which nevertheless is low, cfr. Table VIII below.

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.

Table VIII

Time Oxygenafter concenStirring Tempe

Sample inocutration speed ature Air flow rate Absolute Cells

No. lation (ppm) pH (r.p.m.) (°C) (liters/min.) density per ml. Remarks

1 0^h >10 7.3 200 37.0 2 0.000 0 Before inoculation

2 0^h5^m 10 7.25 0 36.5 2 0.031 0.80×10⁸ After inoculation

3 0^h30^m 7. 3 7.3 0 36.5 2 0.042 1.10

4 0^h50^m 5. 1 7.3 0 36.5 2 0.051 1.35

5 1^h5^m 1. 4 7.3 0 36.5 2 0.061 1.60

6 1^h20^m 0. 1 7.25 0 37.0 2 0.060 1.60

7 1^h35^m 0 7.2 0 37.0 2 0.072 1.95

8 1^h50^m 0 7.2 0 37.0 2 0.080 2.15 9 2^h5^m 0 7.2 0 37.0 2 0.090 2.45

10 2^h20^m 0 7.2 0 37.0 1 0.103 2.80

11 2^h35^m 0 7.2 200 37.0 1 0.123 3.40

12 2^h50^m 0 7.1 200 37.0 1 0.155 4.40

13 3^h5^m 0 7.1 200 37.0 1 0.176 5.05

14 3^h30^m 1. 8 7.0 200 37.0 1 0.191 5.50

15 3^h40^m 1. 8 7.0 200 37.0 1 0.191 5.50

16 4^h5^m 1. 0 6.95 200 37.0 1 0.224 6.55

17 4^h20^m 0 6.9 200 37.0 1 0.235 6.90

18 4^h37^m 0 6.75 200 37.0 1 0.281 8.35

19 4^h50^m 0 6.85 200 37.0 1 0.290 8.65

20 5^h10^m 0 6.85 200 37.0 1 0.340 10.25×10⁸

Table VIII (continued)

Time Oxygen- after concenStirring TemperSample inocutration speed ature Air flow rate Absolute Cells No. lation (ppm) pH (r.p.m.) (°C.) (liters/min.) density per ml, Remarks

21 5^h20^m 0 6.85 200 37.0 1 0.345 10.40×10⁸ 22 5^h35^m 0 6.85 200 37.0 1 0.388 11.70 23 5^h50^m 0 6.85 200 37.0 1 0.365 11.00 24 6^h5^m 0 6.85 200 37.0 1 0.355 10.65 25 6^h20^m 0 6.85 200 37.0 1 0.341 10.25 26 6^h35^m 0 6.85 200 37.0 1 0.338 10.20×10⁸

The results of Table VIII have been used for drawing the growth curve shown in Fig. 3 of the drawings. The abscissa and the ordinate represent the same units as stated in Example 1 for Fig. 1. By a comparison of the curves in Fig. 1 and 2, respectively, and Fig. 3, it is clearly apparent that the growth rate of H. pleuropneumoniae is substantially faster in the CAY-substrate of this invention (Figs. 1 and 2) than in the known CASO--bouillon (Fig. 3) and this is confirmed by calculations according to the formulae (I) and (II) in Example 1 and given below.

From Fig. 3 it will be seen that the exponential growth phase of the bacteria ends at about 5 hours after inoculation and that this phase, as was the case in the fermentation of Example 2 , has two different phases of which phase 1 ends about 3 1/2 hours after inoculation, i.e. about the time where the resumed stirring results in an oxygen concentration above 0 although this period is short.

Calculations made cover samples Nos. 3 to 14 (phase 1) and samples Nos. 14 to 21 (phase 2) and were made as described in Example 1. Again, the basis for the calculation is the points on the curve of Fig. 3 shown with circles. The results are the following

or Tg₁ = 78. 0 minutes and Tg₂ = 119 minutes . These results show that the exponential growth rate of H. pleuropneumoniae bacteria when fermented on the CAY-substrate of this invention is surprisingly faster than the fermentation on CASO-bouillon, viz. approximately twice as fast in phase 1 (Tg = 39.7 and 45.5 minutes versus 78 minutes) and that the growth rate in phase 1 on CASO-bouillon is even slower than the growth rate in phase 2 of Example 2 on CAY-substrate (Tg = 78 minutes versus 75.7 minutes). Besides, the yields on CAY-substrate are surprisingly higher than the yield on CASO-bouillon (26.4 × 10⁸ cells per ml. in Example 1 at the end of the exponential growth phase and 37.2 × 10⁸ cells per ml. in Example 2 versus 10.2 × 10⁸ cells per ml. in Example 3). Thus, the yields on the substrate of this invention are about 2.6 and

3.6, respectively, times the yield on the known substrate.

Example 4

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

2 % w/v solution of aluminium hydroxide. After vigorous stirring overnight (for about 18 hours), the fermentation broth was transferred to a storage vessel, from which it was pumped to a disc centrifuge and separated into an aqueous formaldehyde-containing supernatant which was discarded and a precipitate comprising H. pleuropneumoniae cells and Alhydrogel. The volume of precipitate was about 2 % of the initial broth volume. The precipitate was collected in a vessel. Samples were withdrawn and it was controlled that all bacteria had been killed.

The procedure described in this Example was carried out under aseptic conditions, i.e. the solutions added and the equipment used had been sterilized before coming into contact with the fermentation broth.

Example 5

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.

It will be understood that all of the steps described in this Example were performed under aseptic conditions.

For subsequent use in a vaccination of pigs, it has been found that 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. When adding Alhydrogel it should be taken into consideration that at least the main part of this adjuvant has already been added during the working-up of the fermentation broth described in Example 4. This involves that generally only a minor adjustment of the content of Alhydrogel is necessary and it may even be superfluous to add Alhydrogel together with the "Thiomersal" . The bordetella pertussis suspension which if desired may also be added in the form of B. pertussis biomass and/or extracts thereof, is the ingredient in the H. pleuropneumonia vaccine of this invention which results in the surprisingly superior properties and activities of the vaccine of the invention when used for vaccination of pigs against pleuropneumonia, cfr. the clinical test results reported below.

Neither the B. pertussis vaccine per se (biomass and/or extracts) nor 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, provided that 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. Thus, 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,

Vo. Ill (1975), 409, and complies with the tests mentioned therein.

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.

The various densities were measured on a "Corning Colorimeter 252" at a wave length of 600 nm in the manner described above.

The vaccine is transferred to capped vials in any convenient conventional way. Example 6

By following the procedure described in Example 5, a H. pleuropneumoniae vaccine comprising

H. pleuropneumoniae bacteria 10⁹ cells

Alhydrogel 0.03 ml.

"Thiomersal" 0.1 mg.

Vaccinum pertussis Ph. Eur. (160 I.O.U.) 0.1 ml. Sodium chloride-phosphate buffer (pH 7) to 1 ml.

was prepared.

Clinical Investigations 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. In order to combat pleuropneumonia, the pigs are vaccinated with a H. pleuropneumoniae vaccine of this invention by intramuscular or subcutaneous infection of the vaccine, subcutaneous injection being preferable.

In order to ensure an effective vaccination, 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. However, 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. For pigs of a body weight of above 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.

Example 7

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.

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 10¹⁰ 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)

C. 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 10⁹ 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

Alhydrogel as described in Example 4. The precipitate was diluted with PBS-solution to a concentration of 1 mg/ml. having a content of about 10⁹

H. pleuropneumoniae cells per ml. Dosage 5 mis. (twice).

E. A vaccine of this invention of the composition given in Example 6 and prepared substantially as described in Examples 2, 4, and 5. Dosage 6 mis. (twice).

The results of the vaccination are summarized in Table IX below. Table IX

Vaccine Number of Clinical Changes Remarks by H. p. used test animals symptoms of lungs slaughtering isolated

A 10 2/10^{a )} 0/10^a) 1/10^a) N. D. ^{b )}

B 10 4/10 1/10 0/10 N. D .

C 11 1/11 2/11 1/11 2/2

D 8 4/8 5/8 2/8 1/8

E 6 0/6 0/6 0/6 N . D .

None 3 3/3^{c )} 3/3 3/3 (con trol)

a) Number of animals with symptoms (changes, remarks)/number of test animals. b) Not Determined. c) 1 animal died, 2 were killed when dying.

From Table IX, it will be seen that vaccine E of this invention is superior to the prior art vaccines A to D in providing protection against infections caused by

H. pleuropneumoniae bacteria. 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.

Example 8

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.

The control animals survived until slaughtering, but 2 of the vaccinated animals died before slaughtering. One pig of group L was killed and clearly showed signs of pleuropneumonia. One pig of group E died due to fighting, i.e. for a reason without connection to the present test. Examination of the lungs revealed no signs of pleuropneumonia. The slaughtered animals were examined for the purposes described, in Example 7.

The 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:

C, D, E. As in Example 7. F. 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).

G. As F, except for a content of "Bortavac" as the adjuvant instead of "Quil A". "Bortavac" is a commercial Bordetella bronchiseptica vaccine sold by the producer, Kitasato, Tokyo, Japan.

H. As F, except for a content of "Levoripercol" as the adjuvant instead of "Quil A". "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).

I. As vaccine E, except for a content of "Lymphocyte

Proliferative Factor" (LPF) as the adjuvant instead of Vaccinum pertussis. The LPF was extracted from Bordetella pertussis bacteria as disclosed by S.I. Morse and J.H. Morse, cfr. J. Exp. Med. 143 (1976), 1483-1502.

K. As vaccine E, except for a content of Vaccinum pertussis in an amount of 180 I.O.U.

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. Of the vaccines listed above, vaccines E, I and K are within the scope of the present invention.

The results of the test vaccination of this Example are summarized in Table X.

Table X

Vaccine Number of Clinical Changes Remarks by H. p. used test animals symptoms of lungs slaughtering isolated

C 3 0/3^{a )} 0/3^{a )} 0/3^{a )} N . D . ^{b )}

D 5 0/5 0/5 0/5 N . D .

E 10 0/10 0/10 1/9^{C )} N . D .

F 10 0/10 0/10 0/10 N . D .

G 2 0/2 1/2 1/2 N . D .

H 5 0/5 0/5 1/5 N. D .

a) Number of animals with symptoms (changes, remarks) /number of test animals. b) Not Determined. c) 1 animal died before slaughtering. Table X (continued)

Vaccine Number of Clinical Changes Remarks by H. p. used test animals symptoms of lungs slaughtering isolated

I 5 0/5^{a )} 0/5^{a )} 0/5^{a )} N. D. ^{b )}

K 4 0/4 1/4 0/4 N . D .

L 2 2/2 2/2 1/1^{c )} 1/2

None 2 2/2 2/2 2/2 N . D . (Control)

a) Number of animals with symptoms (changes, remarks)/number of test animals. b) Not Determined. c) 1 animal died before slaughtering.

From Table X it will be seen that clinical symptoms only occurred in aerosol-treated animals and in the control animals. Of the remaining 44 animals, only 2 showed changes of the lungs, and only 3 gave rise to remarks at slaughtering although it could be demonstrated by serological examinations that all animals had been attacked by H. pleuropneumoniae. Thus, 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. Although the various adjuvants tested may seem equally well suited according to Table X, the B. 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. It will be understood that the above description an

Examples are only to be considered as being embodiments of the invention and it will be obvious to those skilled in the art that modifications and variations may be effected therein without departing from the spirit and scope of the invention as defined in the appended claims. In particular, it will be understood that the invention is not restricted to the use of the specifically mentioned strain and/or subculture of H. pleuropneumoniae. Thus, also cultures obtained from, said strain and/or subculture by natural and/or artificial selection and/or mutation as well as cultures of any of the various serotypes are within the scope of the present invention.

Claims

We claim:

1. A vaccine for combating the infectious disease pleuropneumonia in pigs, said disease being caused by the microorganism Haemophilus pleuropneumoniae which comprises H. pleuropneumoniae cells, parts of such cells, extracts and/or metabolism products thereof as the active ingredient and contains adjuvants and a buffer, characterised by a content of a Bordetella pertussis vaccine, a B. pertussis biomass and/or extracts thereof as an adjuvant.

2. A vaccine according to claim 1, characterised by a content of Vaccinum pertussis prepared, identified and tested according to the European Pharmacopoeia, Vol. Ill (1975), 409, as an adjuvant.

3. A vaccine according to claim 1 or claim 2, characterised by a content of Vaccinum pertussis of 16 I.O.U. (international opacity units).

4. A vaccine according to any of the claims 1. to

3, characterised by a content of H. pleuropneumoniae cells, parts of such cells, extracts and/or metabolism products thereof sufficient to provide a density of 20 I.O.U.

5. A vaccine according to any of the claims 1 to

9 4, characterised by a content of about 10 cells of H. pleuropneumoniae per ml.

6. A vaccine according to any of the claims 1 to

5, chaiacterised by having the composition: H. pleuropneumoniae bacteria 10⁹ cells

Alhydrogel 0.03 ml

"Thiomersal" 0.1 mg Vaccinum pertussis, Ph. Eur. (160 I.O.U.) 0.1 ml

Sodium chloride-phosphate buffer (pH 7) to 1 ml.

7. A process for the aerobic fermentation of the microorganism Haemophilus pleuropneumoniae in a liquid fermentation medium comprising assimilable sources of carbon and nitrogen and suitable salts and other nutrient ingredients conventionally used in the fermentation of microorganisms, in particular Haemophilus species, characterised by fermenting in a medium comprising casamino acid, yeast extract, glucose and β-nicotinamide-adenine-dinucleotide (NAD) as essential ingredients.

8. A process accoeding to claim 7, characterised by fermenting in a liquid medium having the composition: Casamino acid 18.0 gs.

Yeast extract 14.0 gs.

Glucose 11.0 gs. NaCl 2.5 gs.

KH₂PO₄ 0.5 g.

MgCl₂·6H₂O 0.1 g.

CaCl₂ (1 % w/v solution) 1.0 ml.

FeSO₄·7H₂O (0.5 % w/v solution, freshly prepared) 2.0 mis. CuSO₄·5H₂O (0.05 % w/v solution) 1.0 ml.

Cystein·HCl (1 % w/v solution) 2.5 mis.

NAD (2.5 % w/v solution) 1.0 ml.

Ion-exchanged water to 1000 mis.

9. A process according to claim 7 or claim 8, characterised by fermenting with stirring of the fermentation medium during the fermentation.

10. A process according to any of the claims 7 to 9, characterised by the fact that a substantially constant pH of the fermentation medium is maintained during the fermentation.

11. A process according to any of the claims 7 to 10, characterised by maintaining a pH of from about 6.0 to about 7.9 in the fermentation medium.

12. A process according to claim 10 or claim 11, characterised by maintaining a pH of from about 6.9 to about 7.5 in the fermentation medium.

13. A process according to any of the claims 10 to 12, characterised by maintaining a pH of about 7.1 to about 7.4 in the fermentation medium.

14. A process according to any of the claims 10 to 13, characterised by maintaining a pH of about 7.3 in the fermentation medium.

15. A process according to any of the claims 7 to 14, characterised by fermenting in a medium having a substantially neutral initial pH.

16. A process according to claim 15, characterised by fermenting in a medium having an initial pH of about 7.2.

17. A process according to any of the claims 7 to 16, characterised by fermenting in a medium having an oxygen concentration of from about 1 to about 30 ppm during the fermentation.

18. A process according to claim 17, characterised by fermenting at an oxygen concentration of from about 8 to about 20 ppm.

19. A process according to claim 17 or claim 18, characterised by fermenting at an oxygen concentration of from about 8 to about 12 ppm.

20. A process according to any of the claims 17 to20, characterised by fermenting at an oxygen concentration of from about 10 to about 12 ppm.

21. A process according to any of the claims 17 to

20, characterised by maintaining the desired oxygen concentration by variation of the stirring speed and/or of the air flow rate.

22. A process according to claim 21, characterised by fermenting at a stirring speed of about 200 to about

600 r.p.m.

23. A process according to claim 21 or claim 22, characterised by fermenting at a stirring speed of about 500 r.p.m.

24. A process according to claim 21, characterised by fermenting at aerobic conditions maintained by using an air flow rate of up to about 0.5 liter of air per minute per liter of fermentation medium.

25. A process according to claim 24, characterised by fermenting at an air flow rate of about 0.15 to about 0.4 liter of air per minute per liter of fermentation medium.

26. A process according to claim 24 or claim 25, characterised by fermenting at an air flow rate of about 0.2 to about 0.3 liter of air per minute per liter of fermentation medium.

27. A process according to any of the claims 7 to 26, characterised by fermenting at a temperature of about 37°C.

28. A process according to any of the claims 7 to 27, characterised by fermenting a subculture of the strain H. pleuropneumoniae ATCC 27089 (NCTC 10976).

29. A process according to claim 28, characterised by fermenting the Subculture 4226 (State Veterinary Serum Laboratory, Copenhagen, Denmark) of the strain H. pleuropneumoniae.

30. A substrate forthe cultivation of microorganisms, in particular suitable forthe cultivation of the microorganism Haemophilus pleuropneumoniae, comprising assimilable sources of carbon and nitrogen and suitable salts and other nutrient ingredients conventionally used in the cultivation of microorganisms, in particular Haemophilus species, characterised by a content of casamino acid, yeast extract, glucose and β-nicotinamide-adenine-dinucleotide (NAD) as essential ingredients.

31. A substrate according to claim 30, in particular suitable for the aerobic fermentation of H. pleuropneumoniae in a liquid fermentation medium, characterised by having the composition: Casamino acid 18.0 gs.

Yeast extract 14.0 gs.

Glucose 11.0 gs.

NaCl 2.5 gs. KH₂PO₄ 0.5 g

MgCl₂·6H₂O 0.1 g

CaCl₂ (1 % w/v solution) 1.0 ml.

FeSO₄·7H₂O (0.5 % w/v solution, freshly prepared) 2.0 mis. CuSO₄·5H₂O (0.05 % w/v solution) 1.0 ml. Cystein · HCl (1 % w/v solution) 2.5 mis.

NAD (2.5 % w/v solution) 1.0 ml.

Ion-exchanged water to 1000 mis.

32. A substrate according to claim 30 or claim

31, characterised by having been. adjusted to a pH of about 7.2.

33. A substrate according to claim 30, in particular suitable for the cultivation of H. pleuropneumoniae on a solid substrate, such as on agar plates, characterised by having the composition:

Casamino acid' 6.0 gs.

Yeast extract 25.0 gs.

Glucose 2.0 gs.

NaCl 2.5 gs.

KH₂PO₄ 0.5 g MgCl₂·6H₂O 0.1 g.

CaCl₂ (1 % w/v solution) 1.0 ml.

FeSO₄·7H₂O (0.5 % w/v solution, freshly prepared) 0.5 ml.

Cystein·HCl (1 % w/v solution) 0.8 ml.

Agar-agar 16.0 gs. NAD (2.5 % w/v solution) 1.0 ml.

Ion-exchanged water to 1000 mis.

34. A substrate according to claim 33, characterised by having been adjusted to a pH of about 7.2.