NL8020132A - A VACCINE FOR THE FIGHT AGAINST PIG PURPOSEUMONIA IN PIGS, A METHOD AND A SUBSTRATE FOR THE AEROBIC FERMENTATION OF HAEMOPHILUS PLEUROPNEUMONIAE. - Google Patents

Description

N.O. 29.59¾ -1-t, ÜÜÖ 132 ^

A vaccine for the control of pleuropneumonia in pigs and a method and a substrate for the aerobic fermentation of Haemo-philus pleuropneumoniae .___

Background of the invention.

In the early 1960s, Haemophilus-like bacteria were isolated in Britain, California and Argentina from pig herds affected by pleuropneumonia. It was the bacterium Haemophilus parahaemolyticus - or Haemophilus pleuropneumoniae as the Argentinian strain was called - that caused the disease pleuropneumonia. Attacks of the disease have since been reported in many countries, including Denmark, see R. Nielsen, Nord. Fat. Med. 22 (1970), 2 ^ 0-2 ^ 5.

The disease has a very acute course and is often accompanied by a high mortality rate and, since it occurs quite frequently in many pig herds, a number of efforts have been made in an attempt to develop a vaccine that would protect the pigs against the disease. A common feature of this vaccine. is their use of killed cells of H. pleuropneumoniae and in most cases also an addition of an additive.

Vaccination tests have been performed under field conditions, see R. Nielsen, Nord. Fat. Med. 28 (1976), 337-3 ^ + 8. In these experiments, 6 or 2b hour old agar plate cultures of H. pleuropneumoniae were used as an antigen. The cells were killed by formaldehyde 10 and the vaccine contained 10 cells per ml in phosphate buffered saline (physiological) (hereinafter PBS for brevity) containing 0.2% formaldehyde and to which an additive had been added. The vaccine was administered as subcutaneous injections of 2 times b ml or 2 times 2 ml with an interval of 10 days. The first vaccination was performed when the pigs were 9 weeks old. Two weeks after the second vaccination, the pigs were infected with 10 H. pleuropneumoniae bacteria. As additives, 15% Alhydrogel (aluminum hydroxide gel) and Freund's incomplete additive, respectively, see J. Freund, Am. Rev. Microbiol. 1_ (19 ^ 7), 291.

The results of these vaccination trials are summarized in the following table.

Table I.

Age of Antigen_Additive_Protection (%) 35 6 hours 15% Alhydrogel 6? __________________ Freund's unfull ._90_ 8020132 ^ -2- v

Table I (continued)

Age of Antigen Additive Protection ($) 2 hours 15 $ Alhydrogel 25 Freund's incomplete 6-5 It should be added that the use of Freund's incomplete additive gave rise to granulomas at the injection site.

Attempts have also been made to obtain protection by using 20 hours of cultures killed by formalin as the antigen and Alhydrogel and Freund's complete additive as additives, respectively. The injected dose was 2 times 5 ml containing 9 bacteria per ml. The first injection was performed intramuscularly on 50 day old pigs and the second injection 1 week later. The pigs were infected one week later. The result was clear evidence that the vaccine had a protective effect, but no clear evidence that one additive had advantages over the other.

E. Scholl et al., See Proc. Int. Pig Vet, Soc. Congress, Ames, Iowa, U.S.A. (1976), have used heat-killed cultures of H. pleuro-20 pneumoniae as antigens and have been vaccinated by two methods: 1) with increasing doses, i.e. 0.5 1 and 3 ml and 2) with 2 times 5 ml. All injections were performed at 2-week intervals. Serological examination of blood samples showed that repeated injections of small doses resulted in antibodies in the blood, however no sensitized lymphocytes, whereas injections of higher doses resulted in sensitized lymphocytes, but no antibodies in the blood.

The same workers, see Proc. Int. Pig Vet. Soc. Congress, Zagreb, Yugoslavia (1978), have used cultures of H. pleuropneumoniae killed with formalin at concentrations of 10 bacteria per ml. The vaccination doses and the vaccination methods were the same as in the previously mentioned work, however in this later work a group of placebo pigs was included and the animals were brought into a chronically infected pig herd. Clinical signs were observed and after slaughter, the lungs and chest cavity were examined. The results showed a good effect of the vaccination, but no differences between the two vaccination methods.

M.F. de Jong, see Proc. Int. Pig Vet. Soc. Congress, Zagreb, Yugoslavia (1978), has performed vaccination trials with 2b hours H.

f0 pleuropneumoniae cultures, to which was added an incomplete additive on mineral oil base 8020132 v -3- sis. For each dose, 20 mg (wet weight) of antigen were used and two injections were made at 3-week intervals, the first vaccination when the pigs were 10 weeks old. Three weeks after the second vaccination, the pigs were challenged with 16 or 2b hours of culture. With 2b hours of challenge cultures, the pigs became ill, but recovered within 1 week. On the other hand, the pigs died stimulated with a 16-hour challenge culture within 36 hours. As will be known to the skilled person, the challenge involves an experimental infection of the test animals with living cells of the infecting micro-organism.

M.F. de Jong, l.c., then used 6 hours of culture for both vaccination and stimulation. The pigs were challenged with different amounts of cells and the mortality rate depended on the number of cells used. In addition, pigs, 13 vaccinated with 6 hours of culture, were challenged with 16 hours of culture and 50% of the pigs survived. One week later, the surviving pigs were infected with a 6 hour culture and now they did not even show any symptoms of disease.

The work with test vaccinations, as mentioned above, clearly shows that it is necessary to add an additive to a vaccine containing H. pleuropneumoniae antigens in order to obtain a satisfactory effect of the vaccination. The products most commonly used as additives are Alhydrogel and Freund's additive (complete or incomplete).

Of these products, Freund's additive has been the most effective so far, but as also noted, see R. Nielsen, loc. cit. (1976), this results in the formation of granulomas at the injection site and is therefore considered unsuitable. Therefore, the problem of providing an additive which is effective but does not affect the animals has not been solved by the aforementioned test vaccinations.

Another problem not solved is the preparation of an antigen for the production of the vaccine. This problem lies in the fact that H. pleuropneumoniae has a rather poor growth on the previously used and classic substrates, which have very limited limits. the amount of vaccine that can be produced.

Therefore, J. Nicolet, see Zbl. Bakes. I Abbot. Orig. 216 (1971), ^ 87- ^ 95, in his seriological studies uses a substrate containing PPLO agar (see DIFCO-Manual IX, 1953i Catalog No. b '- 2 - PPLO = pleuropneumoniae-like organisms) to which 8020132 -4- * 0.1% glucose, 2.5¾ yeast extract, 5¾ horse serum and 5mg / liter NAD (β-nicotineamide-adenine dinucleotide) are added. The same substrate 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., see Int. J. Syst. Bacteriol, 28 (1978) »20-26, has conducted taxonomic studies of H. pleuropneumoniae, in which the strains were grown on a substrate that neutralized 1% bacteriological peptone (" Oxoid ")» 10 1% yeast extract, 1% glucose, 10 mg / liter NAD, and a saline solution as described by LV Holdeman and W.E.C. Moore in Anaerobe Laboratory Manual, 3rd ed., Virginia 15 Polytechnic Institute, Anaerobe laboratory, Blacksburg (1975) ».

Some other workers have grown 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 effective control of pleuro-pneumonia in pigs, also providing a new and improved additive for such a vaccine without the side effects associated with the known additives.

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 the present invention to enable an effective vaccine to be in amounts sufficient for any desirable extended vaccination.

A further object of the invention is to provide a new and improved substrate for growing H. pleuropneumoniae to achieve growth of the microorganism. which is considerably better than when grown on known substrates.

Yet a further object of the invention is to provide a method of controlling pleuropneumonia in pigs by administering the vaccine of the present invention.

These and other objects are achieved by providing a vaccine containing cells of Haemophilus pleuropneumoniae, parts 40 of such cells, extracts and / or metabolism products thereof 8020132, -5- obtained by culturing a strain of H. pleuropneumoniae in a liquid culture based on the substrate described by SM Cohen and M.W. Wheeler in Am. J. Publ. Hlth. 36 (19 ^ 6), 371-376, which vaccine also contains as an additive aluminum hydroxide gel and / 5 Freund's incomplete or complete additive and / or Bordetella pertussis vaccine, biomass and / or extracts thereof, as well as a sterile buffer solution as diluent .

Detailed description of the invention.

The bacterium used to produce the vaccine of the present invention is a strain of Haemophilus pleuropneumoniae, which is a known microorganism, deposited in various culture collections, see below, and available there.

Investigations have shown that at least 6 different types of H. pleuropneumoniae exist, see M. Kilian et al., Loc. 15 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 provides a general overview of the micro-organism's properties.

Of the various serotypes of H. pleuropneumoniae described in the preceding literature reference, some have been deposited, such as serotype 1 in the American Type Culture Collection, Rockville, Md., USA, under No. ATCC 27088, serotype 2 under No. ATCC 27089, and serotype 5 under number ATCC 27090. In addition, serotype 2 has been deposited in the National Collection of Type Cultures, Colindale, London, 25 UK, under no. NCTC 10976 and these three serotypes have all been deposited in the Czechoslovak Collection of Microorganisms, Brno, Czechoslovakia, under Nos. CCM 5869, CCM 5870 and CCM, respectively

5871.

In addition, M. Kilian et al., Loc. cit. (1978) serotypes-30 pen referred to as serotypes * f, 5 and NT. However, no deposit numbers are given.

Applicants' own investigations have confirmed that H. pleuropneumoniae bacteria are very small Gram-negative immobile rods, which can take a number of different forms depending on the culture substrate used and the environmental conditions.

Thus, on agar plates, they are usually small coccoid or short bars, while in liquid cultures they can range from small coccoid bars to longer thin or thick bars, with the exception of bO even (which is quite rare) to pleomorphic filament forms.

8020132 -6-

Cultured on agar plates on the substrate of the present invention (composition, see Table III) below), H. pleuropnea-moniae bacteria are formed as small coccoid rods and the individual colonies are up to 3 mm in diameter after 48 hours at 37 ° C . Fermented in the nutrient broth of the present invention (composition see Table IV below), H. pleuropneumoniae bacteria are short rods, often in shorter or longer chains.

Biochemically, H. pleuropneumoniae bacteria behave as described by M. Kilian, J. Gen. Microbiol. 93 (1976), 10 9-62, having the following characteristic properties, which together make them different from other Haemophilus species: V factor-dependent, capable of synthesizing porphyrin from delta-aminolevulinic acid, producing urease and capable of to ferment mannitol, xylose and deoxyribose.

More specifically, a strain of H. pleuropneumoniae isolated from a pig with pleuropneumonia has been used. This strain was of serotype 2, see R. Nielsen, loc. cit. (1976), and was transferred to Statens Veterinaere Serum Laboratory (State Veterinary Serum Laboratory), Copenhagen, Denmark, where it was designated Subculture 4226.

The substrate used in the practice of the present invention is based on the substrate described by S.M. Cohen and M.W. Wheeler, loc. cit., with the composition given in Table II below.

Table II.

Casamino acid *), technically 10.0 g

NaCl 2.5 g κη2ρο ^ 0.5 g

MgCl2.6H20 0.4 g of soluble starch 1.5 g

CaCl2 (1% solution) 1.0 ml

FeS0 7 .7H 2 O (0.5 # solution) 2.0 ml

CuSO ^ ^ I ^ O (0.05 # solution 1.0 ml cysteine.HCl (1 # solution) 2.5 ml 35 yeast dialysate 50.0 ml distilled water to 1 liter pH adjusted to 7.2-7.3.

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

For the purposes of the present invention, this substrate is modified to enhance the growth of H. pleuropneumoniae and provide a substrate suitable for the preparation of an H. pleuropneumoniae vaccine in sufficient amounts for a desired comprehensive vaccination. Two different modifications are used for culturing on agar-5 plates and fermentation in a liquid medium, respectively.

For agar plates, a substrate containing casamino acid, yeast extract, glucose and β-nicotineamide adenine dinucleotide (hereinafter referred to as NAD) is used as essential ingredients. In addition, this substrate should contain suitable salts and other nutritional ingredients commonly used in the cultivation of microorganisms, especially Haemophilus species. A particularly preferred substrate has the composition as given in Table III below.

Table III.

15 Substrate for agar plates.

casamino acid, technical (Difco Manual) 6.0 g yeast extract, technical (Difco Manual) 25.0 g glucose (Pharmacopoea Nordica) 2.0 g

NaCl 2.5 g 20 ΚΗ2Ρ0 ^ 0.5 g

MgCl2.6H20 0.1 g

CaCl2 (1 w / v% solution) 1.0 ml

FeS0 ^ .7H20 (0.5 w / v solution, freshly prepared) 0.5 ml cysteine.HCl (1 w / v solution) 0.8 ml 25 agar (Difco Manual) 16, 0 g NAD (2.5 w / v solution) 1.0 ml (Sigma Chemical Co., St. Louis, Mo., USA) water to 1000 ml.

This substrate is recovered in the following manner: Casamino acid, yeast extract, glucose, NaCl, KH2PO2 and MgCl2i6H20 are dissolved in 950 ml of ion exchanged H20. The pH is adjusted to 7.2 by the addition of 1 N NaOH. CaCl2, FeSO7.7H2O, cysteine.HCl and agar agar are added and the volume adjusted to 1000 ml by the addition of ion-exchanged water.

The solution is heated to boiling (to dissolve the agar), and autoclaved at 121 ° C and 2 atmosphere pressure for 15 minutes. After cooling to 50 ° C, the NAD is added and the substrate is poured into Petri dishes.

The substrate of the present invention for use in the kO fermentation in a liquid medium also contains casamino acid, yeast extract, glucose and NAD 8020132-8, as essential ingredients. This substrate should also contain suitable salts and other nutritional ingredients commonly used in the fermentation of microorganisms, especially Haemophilus species. A particularly preferred substrate has the composition shown in Table IV below.

Table IV,

Substrate for fermentation in liquid medium, casamino acid, technical (Difco Manual) 18.0 g 10 yeast extract, technical (Difco Manual) 14.0 g glucose (Pharmacopoea Nordica) 11.0 g

NaCl 2.5 g ΚΗ2Ρ0 ^ 0.5 g

MgCl 4 H 1 O 0.1 g 15 CaCl 2 (1 w / v solution) 1.0 ml

FeSO7.7H2 O (0.5% w / v solution, freshly prepared) 2.0ml

CuSO 2 + .5H 2 O (0.05 wt.% W / v solution) 1.0 ml cysteine HCl (1 w / v% solution) 2.5 ml NAD (2.5 w / v% solution) solution (Sigma Chemical Go.) 1.0 ml of water to 1000 ml.

This substrate, which is termed CAY substrate for brevity, is prepared in the following manner:

Casamino acid, yeast extract, glucose, NaCl, KH2PO2 and MgCl2.6H2 O are dissolved in 950 ml of ion-exchanged water. The pH is adjusted to 7 * 2 by the addition of 1 N NaOH. CaCl2, FeS0 ^ .7H20, CuS0 ^ .5H20 and cysteine.HCl are added, the volume is adjusted to 1000 ml by the addition of ion-exchanged water and the solution is autoclaved for 15 minutes at 121 ° C and a pressure of 2 atmosphere. After cooling, NAD is added -50 and the substrate is ready for use.

Both substrates described in Tables III and IV contain assimilable sources of carbon and nitrogen which can be usefully utilized by the microorganism H. pleuropneumoniae. An abundant and dense growth of the organism is obtained on both substrates.

The fermentation method is as follows.

The bacteria strains are stored in a lyophilized state in ampoules prepared by one of the methods described below. Storage is carried out in a refrigerator at 5 ° C.

Method A. The bacteria were grown at 37 ° C for 18 8020132-9 hours on agar plates with a substrate having a composition as set forth in Table III. The cells were harvested by the addition of 1 ml PBS (composition given in Table V below) and subsequent cell deletion. The cells were transferred to 5 ampoules, which were immediately cooled to -25 ° C and then transferred to a lyophilizer and lyophilized.

Table V.

Composition of PBS.

NaCl 8.0 g 10 KOI 0.2 g

Na ^ PO ^ 1.15 g of ΚΗ2Ρ04 0.2 g of distilled water to 1 liter.

The salts are dissolved in distilled water and autoclaved for 15 minutes at 121 ° C and a pressure of 2 atmospheres. The pH is 7 »3.

Method B. This method was similar to Method A, but 1 ml skim milk was used for each plate instead of PBS. The skim milk was heat treated by heating to 100 ° C for 15 minutes on three consecutive days.

Method C. The bacteria were grown on a water bath in the CAY substrate for 6 hours, see Table IV. The fully developed bacterial suspension 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 recovered 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 daily inoculation on fresh agar plates containing the substrate of Table III. The plates are incubated in an incubator at 37 ° C.

A pre-culture is prepared by propagation of the bacteria. This is performed by inoculation from an agar plate into a flask containing 50 ml of the liquid CAY substrate. The flask is incubated in an incubator at 37 ° C for 8 hours. 10 ml of medium is then withdrawn for inoculation of the second pre-culture.

The second pre-culture is 500 ml of the liquid CAY substrate ko in a 1 liter conical flask, which is incubated on a water bath with continuous shaking. The temperature is 32 ° C and the incubation period is 18 hours.

The second pre-culture 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, ie by dissolving casamino acid, yeast extract, glucose, NaCl, IQ ^ PO ^ and MgCl2 »eH20 in ion-exchanged water, adjusting the pH to about 7i2, the adding the remaining ingredients except the NAD, autoclaving the environment at a temperature of at least about 121 ° C and a pressure of at least about 2 atmospheres for at least 15 minutes, cooling to a temperature of no more than 37 ° C, adjusting and resetting the pH to about 7 »2 and adding NAD.

The fermenter is then inoculated with the second pre-culture of H. pleuropneumoniae obtained as described above. During the fermentation, a temperature of preferably about 37 ° C is maintained and an air flow through the environment is used to maintain an appropriate oxygen concentration in the environment. This can be done in part by controlling the airflow rate and in part by controlling the speed of the stirrer of the fermenter, taking into account that these parameters are mutually dependent, as far as increasing one 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 can range from about 1 to about 30 ppm, the most suitable concentration being about 8 to about 20 ppm, especially about 8 to about 12 ppm, more especially about 10 to about 12 ppm. The air flow rate and agitation speed necessary to maintain such an oxygen concentration will, of course, depend upon the size, equipment and arrangement of the fermenter, however, it has been found that an air flow rate of up to about 0.5 liters of air per minute per liter of fermentation medium, in particular about 0.15 to about 0.1 liter, more particularly about 0.2 to about 0.3 liters of air per minute per liter of fermentation medium is most suitable in a fermenter with a JfO capacity of approximately 20 liters. For this fermentor 8020132 -11-, a suitable stirring speed is about 200 to about 600 revolutions per minute, especially about 500 revolutions per minute.

It should be emphasized that these parameters are not critical to the practice of the present invention, and for any fermentor, one skilled in the art will be able to control the parameters to maintain the desired oxygen concentration.

It is preferable to maintain a substantially constant pH during fermentation. 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 particularly from about 7 to about 7.4, preferably at about 7 »3. This can be accomplished by the optional addition of a base, an aqueous solution of sodium hydroxide being suitable.

It is not preferable to add an anti-foaming agent, however it may be necessary to do so. Any conventional anti-foaming agent for use in fermentation processes is suitable for this purpose, in particular a silicone anti-foaming agent.

The fermenter is preferably provided with means which allow an automatic addition of a pH controlling base and / or an anti-foaming agent during the fermentation. However, the addition can also be carried out manually if necessary.

During the fermentation, intermittent samples are taken and used to determine the cell density. The density was measured with a "Corning Colorimeter 252" at a wavelength of 600 nm and expressed as the number of colony forming units (CFU) by a reference standard curve. The purpose of this density measurement is to draw a growth curve which makes it possible to allow the interruption of fermentation at the most appropriate time for the subsequent production of a vaccine, ie when the phase in which the bacteria show exponential growth has passed.

When the fermentation is complete, a concentrated form aldehyde solution and an Alhydrogel solution are added to the fermentation medium and after stirring for a considerable time, for example overnight, the suspension is subjected to centrifugation. formaldehyde containing supernatant and a precipitate containing H. pleuropneumoniae cells and Alhydro-kO gel. The filtrate is collected in a sterile reservoir.

8020132 -12-

A sample is taken to check that the bacteria have been killed.

The following examples further illustrate the benefits obtained by growing H. pleuropneumoniae on the CAY substrate of the present invention.

Example I.

This example shows the fermentation of Haemophilus pleuropneumoniae, strain ATCC 27089, subculture 4226 on the CAY substrate of the present invention.

The fermenter was a "Biomat E 20" (Moeller and

Jochumsen, Vejle, Denmark) with a capacity to contain 20 liters of substrate in each fermentation and provided with means for stirring, which means allow a manual variation of the stirring speed, an instrument that continuously shows the stirring speed 15 (rpm) , means for supplying sterile air, means for a continuous measurement and registration of the oxygen concentration of the fermentation medium, means for a continuous measurement and automatic control of the temperature at a predetermined value, means for a continuous measurement and registration 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 fermenter and for the automatic addition of antifoaming agent, if necessary, and means for withdrawing samples during the fermentation.

The fermenter was charged with 17 liters of ions from exchanged water and stirring was started. Then 306 g cas amino acid, 258 g yeast extract, 18 g of glucose, 42.5 g of NaCl, 8.5 g of KH2 PO2 and 1.7 g of MgCl2.6H2 O were added. The pH was adjusted to 7 2 by the addition of a 1 N NaOH solution and 17 0 ml of a 30 1 w / v solution of CaCl 2, 34.0 ml of a 0.5 w / v. Solution of FeSO5.5H2O (freshly prepared), 17.0 ml of a 0.05 w / v solution of CuS0.5 H20 and 42.5 ml of a 1 w / v solution of cysteine HCI were added. The fermenter was closed, autoclaved for 15 minutes at 121 ° C and 2 atmospheres pressure, then cooled to 36 ° C. Then, 17 ml of a 2.5 w / v solution of NAD was added. This method resulted in a fermentation medium with the composition given in Table IV and with a pH of 7.45 »

The fermenter was inoculated with 600 ml of a second 40 pre-culture of H. pleuropneumoniae prepared from a lyophilized 8020132-13 bacterial stock and grown as described above. The fermentation period was 7 hours 50 minutes and samples were extracted intermittently and used to determine the absolute cell density of the fermentation medium. The density was measured on a "Corning Colorimeter 252" at a wavelength of 600 nm and then converted to cells per ml using a standard reference and a conversion table, which allows to draw a growth curve and determine when the exponential growth phase of the bacteria had come to an end. This fermentation was performed primarily as an experimental fermentation and was therefore allowed to proceed in a somewhat "abnormal" manner, which meant that the pH was not adjusted during the fermentation and that no anti-foaming agent was added.

The results obtained from the analysis of the samples are given in Table VI below.

It has been found that the oxygen concentration, which should preferably be 8-10 ppm, decreased drastically during the fermentation, especially in the exponential growth phase. This was counteracted by partly increasing the airflow rate and partly the stirring speed. It also appears that the pH decreased to a final pH of 5 * 30.

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8020132 -16-

The results in Table VI have been used to draw the growth curve indicated in Figure 1 of the drawings. In the figure, the abscissa shows the time after inoculation and the ordinate shows the number of cells per ml of fermentation medium. It is noted that the ordinate is represented 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 must be stopped at this time, when the medium is intended for use in the preparation of a vaccine. However, since this fermentation was experimental fermentation, it was continued to the stationary growth phase.

Fig. 1 of the drawings also makes it possible to calculate the time (Tg) necessary to double the bacterial content in the fermentation medium during the exponential growth phase. Tg is given by the formula log 2

Tg = -1- (I)

/YOU

where ^ u is represented by the formula log 0 - log C

/ u -T ~ - (II) where C ^ is the number of cells per volume unit at time 20 after inoculation and the number of cells per volume unit at time T ^ is after inoculation.

In Figure 1, the exponential growth phase is represented by the straight line portion of the curve. Therefore, it appears that this phase extends from no later than 1 hour to about 5 hours after inoculation. Use of this part of the curve (the results marked with a circle in Fig. 1, i.e. sample nos. 6 to 18) for a calculation according to formula (II) shows that 3.27 + 0.92 / U = 3 1- = 1.05 where T_ and T. are expressed as hours and the common factor of 10 ° is ignored in the calculation of log 0o and log CL.

6 c. 6 I

According to formula (I), this means that Tg = -f-'-jp- = 0.660 hours which corresponds to 39.6 minutes.

8020132 -17-

Example II.

This example shows a "normal" fermentation, which means that the pH is continuously and automatically controlled at about 7.2, anti-foaming agent is added automatically if necessary, a more vigorous stirring is maintained and the oxygen concentration is continuously a relatively high level is maintained.

The fermenter was identical to the fermenter used in Example I. The volume of the fermentation medium was about 20 liters, of which the inoculation medium provided 0.5 liters, the remainder consisting of 19 liters of ion-exchanged water containing casamino acid, yeast extract, glucose and salts in amounts, which are a CAY substrate of the relative composition given in Table IV. The filling of the fermenter, the autoclave treatment, the cooling and the subsequent addition of NAD is carried out as described in example I.

When the inoculation medium was added, the fermentation was started and continued for 7 hours and 30 minutes. The inoculation medium was 500 ml of a second pre-culture of H. pleuropneumoniae, strain ATCC 27089, subculture * f226, grown in the manner described above.

During the fermentation, the pH was maintained at a nearly constant value of about 7.2 by the automatic addition of 1 N NaOH solution, if necessary. The stirring treatment was carried out at 500 rpm. and the oxygen concentration was maintained at about 8 to about 12 ppm. This was accomplished by increasing the airflow rate if necessary. An anti-foaming agent with the trade name "Silicone RS Antifoam C. DAK" was added automatically if necessary.

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

The results of the fermentation are summarized in Table VII.

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The data reported in Table VII has been used to draw the growth curve shown in Figure 2 of the drawings. The abscissa and the ordinate have given the meanings for Fig. 1, see Example 1. Fig. 2 shows that fermentation 5 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, while a slight decrease in the slope of the curve occurred at about 3 hours and 30 minutes after the inoculation.

The measurements indicated by circles in fig. 2 have been used for calculations according to formulas (I) and (II) of example I, partly for the initial phase (phase 1, sample numbers 3 to 13) and partly during the last period (phase 2 sample nos. 13 to 27) of the fermentation. The units used in the calculations of Example 11 were also used in these calculations, giving the following results.

u = = 0.914 h ”1, / 3.25 - 0.8

Tg = M2L = 0.758 hours 0.914 which equals 45.5 minutes.

20 -h = h§.2., - 1, t79. = 0.549 h "1.7 2 7.5-4

Tg = 1.26 hours 0.549 which is equal to 75.7 minutes.

A comparison of the results obtained according to Examples I and II will show that although the "abnormal" fermentation process of Example I results in the lowest initial Tg, which means faster growth of the bacteria in the fermenter and consequently a shorter fermentation period before the broth is ready for use for the production of an H, pleuropneumoniae vaccine, the method also results in a rapid transition in the stationary growth phase, viz. after a fermentation period of about 5 hours, while the "normal" fermentation results in an extended period of the exponential growth phase of the bacteria, viz., more than 7.5 hours. More importantly, the "normal" fermentation also results in a greater yield of cells per 8020132 ... * -21 volume unit at the time when the broth is ready for vaccine production, i.e. more than 37 x 10 cells per ml compared to g about 27 x 10 cells per ml in Example I (Sample No. 18, ending the exponential growth phase). Therefore, from a commercial point of view (vaccine production), the "normal" fermentation process of Example II is preferred, because the greater time consumption is more than counterbalanced by the greater yield of cells, which can be usefully utilized in the subsequent vaccine production. .

Example III.

This example is a comparative example demonstrating 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 the present invention.

The fermenter was identical to the fermenter used in Examples I and II.

The substrate was a "CASO broth Merck" commercially described as No. 5 ^ 78 in "Handbook of Microbiology" published by E. Merck, Darmstadt, Federal Republic of Germany, containing 6 mg of NAD per liter of broth have been added. The fermenter was charged with 17 liters of CASO broth, autoclaved and cooled to 37 ° C as described in Example I, then NAD was added. The starting pH was 713.

As an inoculation medium, 600 ml of a second pre-culture of H. pleuropneumoniae ATCC 278-991 subculture 4226 was added.

This pre-culture was grown in the manner described above. A temperature of 37 ° C was maintained during the fermentation. The pH was adjusted manually and allowed to decrease to a final pH of 6.85. The initial oxygen concentration was 10 ppm, but it dropped very quickly to 0, in part because the air flow rate is low and in part because the stirring was stopped immediately after inoculation and did not resume until 1 hour after an oxygen concentration of 0 was reached.

It is therefore characteristic of this fermentation that it must take place under nearly anaerobic conditions in order to obtain an acceptable yield, which is nevertheless low, see Table VIII below.

The fermentation period was 6 hours and 35 minutes, during which time samples were taken and analyzed in the manner 8020132 -22- • Η Η Μ>

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The results of Table VIII were used to draw the growth curve shown in Figure 3 of the drawings. The abscissa and the ordinate represent the same units as reported in Example I for Figure 1. By comparing curves 5 in Figures 1 and 2 and Figure 3, respectively, it is very clear that the growth rate of H. pleuropneumoniae is significantly faster in the CAY substrate of the present invention (fig. 1 and 2) than in the known CASO broth (fig. 3) and this is confirmed by calculations according to formulas (I) and (II) in example I and 10 below.

It can be seen from Fig. 3 that the exponential growth phase of the bacteria ends at about 5 hours after inoculation and that, as was the case with the fermentation of Example II, this phase has two different phases, phase 1 about 3.5 hours after inoculation 15 ends, that is, approximately the time when the resumed stirring treatment results in an oxygen concentration above O, although this period is short.

The calculations made cover sample nos. 3 to 1k (phase 1) and sample nos. K to 21 (phase 2) and were performed as described in Example I. Again, the points of the curve of Fig. 3 * are indicated by circles, the basis for the calculation. The results are the following: Λ = 3.5 - 0.5 = h 2,3 2.3 ^ - 1.70, / U U — 0,5 50 0.550 h, 5 5.33 - 3.5 25 Tg = 1.30 h 0.535

Tg = 2 ^ 22 = 1.98 h, 0.350 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 the present invention, is surprisingly faster than the fermentation on CASO broth, i.e. about twice as fast at phase 1 (Tg = 39.7 and ^ 5.5 minutes at 78 minutes) and that the growth rate in phase 1 on CASO broth is even slower than the growth rate in phase 2 of Example II on CAY substrate (Tg = 78 35 minutes at 75, 7 minutes).

8020132 -25 "

In addition, the yields on CAY substrate are surprisingly greater than the yield on CASO broth (26 x 10 cells per ml in Example I at the end of the exponential growth phase and 8 8 37 i2 x 10 cells per ml in Example II against 10.2 x 10 cells per 5 ml in Example III). Therefore, the yields on the substrate of the present invention are about 2.6 and 3i6 times the yield on the known substrate, respectively.

Example IV.

After a fermentation period of 7 * 5 hours, the fermentation described in Example II was stopped by the addition of 60 ml concentrated formaldehyde solution of 600 ml Alhydrogel. The added Alhydrogel was a 2% w / v solution of aluminum hydroxide. After vigorous stirring overnight (about 18 hours), the fermentation broth was transferred to a storage reservoir, from which it was pumped to a disc centrifuge and separated into an aqueous formaldehyde containing supernatant, which was removed and a precipitate containing H. pleuropneumoniae. cells and Alhydrogel. The volume of the precipitate was about 2% of the initial broth volume. The precipitate was collected in a reservoir. Samples were taken and all bacteria were checked to be killed.

The method described in this example was performed under aseptic conditions, ie the added solutions and equipment used were sterilized before contacting the fermentation broth.

Example V.

The precipitate, collected as described in Example IV, was used to produce an H. pleuropneumoniae vaccine-j. A vaccine was obtained by diluting the precipitate with a phosphate buffered saline (physiological) (PBS) solution of the preparation indicated in Table V to approximately the desired density of the vaccine. Then "Thiomersal" [sodium 2- (ethyl mercury) -thio) benzoate and optional Alhydrogel were added and the precipitate, PBS solution, "Thiomersal" and Alhydrogel were suspended in a sodium chloride phosphate buffer with a pH of 7 to 20 minutes and a sample was withdrawn for analysis. in this analysis, the density of the vaccine was adjusted to the desired value, then a Bordetella pertussis suspension (a B. pertussis vaccim) was added and the stirring treatment was continued for 30 minutes, the density was controlled and the vaccine was filled 80 2 D13 2 -26- in sealed bottles.

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

For a subsequent use in a vaccination of pigs 5, it has been found that a suitable vaccine density *, about 20 I.O.U. it is preferable that the precipitate of Example IV be diluted with PBS solution to about this value and that the final density of the vaccino be controlled at a level of 20 I.O.U. before the addition of the B. per-10 tussis suspension. It has also been found that a suitable concentration of the additive "Thiomersal" is about 0.01% w / v and that the additive Alhydrogel should preferably be present at a concentration of about 3% by volume. When adding Alhydrogel, it should be considered that at least the major part of this additive has already been added during the work-up of the fermentation broth described in Example IV. Bit implies that generally only a minor adjustment of the Alhydrogel content is necessary and that it may even be unnecessary to add Alhydrogel together with the "Thiomersal".

The Bordetella pertussis suspension, which may also be optionally added in the form of B. pertussis biomass and / or extracts thereof, is the constituent in the H. pleuropneumoniae vaccine of the present invention, resulting in the surprisingly better properties and activities of the vaccine of the invention, when used for vaccinating pigs against pleuropneumonia, see clinical trial results as set forth below.

Neither the B, pertussis vaccine as such (biomass and / or extracts) nor the method of its production forms part of the present invention, which means that any such vaccine is useful as an additive in the H. pleuropneumoniae vaccine of the invention, provided that the B. pertussis vaccine, as well as the final vaccine of the present invention, meets all the requirements of vaccines according to national and / international standards. Therefore, the preferred B. pertussis vaccine for use in the production of an H. pleuropneumoniae vaccine of the present invention is a Vaccinum pertussis, which has been prepared, identified and tested as described in European Pharmacopoeia, Part III (1975) i. ^ 09 and satisfies the tests stated therein.

JfO An appropriate amount of the additive B. pertussis suspension 8020132-27 suspension in the H. pleuropneumoniae vaccine of the invention is approximately 16 I.O.U. vaccine-:.; which can be conveniently obtained by adding 100 ml of B. pertussis suspension with a density of 160 I.O.U. to 1 liter of suspension obtained by mixing the precipitate of Example IV, PBS solution, "Thiomersal", Alhy drier when used and sodium chloride phosphate buffer.

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

10 The vaccine. is transferred to capped vials in any suitable conventional manner.

Example VI.

Following the method described in Example V, an H.

pleuropneumoniae vaccine-j, which contains * 9 15 H. pleuropneumoniae bacteria 10 cells

Alhydrogel 0.03 ml "Thiomersal" 0.1 mg

Vaccinum pertussis Ph. EUR. (160 I.O.U.) contains 0.1 ml of sodium chloride phosphate buffer (pH 7) to 1 ml of 20.

Clinical Investigations.

The vaccine of the present invention has been shown to be effective in controlling pleuropneumonia in pigs exposed to infection by the micro-organism Haemophilus pleuro-pneumoniae and is therefore indicated for the prophylactic treatment of pigs, especially seronegative pigs, before she. be transferred to an environment already infected with H, pleuropneumoniae or where there may be a risk of such infection. In order to combat pleuropneumonia, the pigs are vaccinated with an H. pleuropneumoniae vaccine of the present invention by intramuscular or subcutaneous injection of the vaccine, with subcutaneous injection being preferred.

In order to ensure effective vaccination, the vaccine of the present invention is administered twice with an appropriate period between injections, generally not less than 3 weeks. The dose administered will, of course, depend on the body weight of the pigs, the strength of the vaccine, the risks of side effects and the nature of such effects, if any, and other factors that are usually considered when vaccination is performed, except of the active ingredient in the vaccine. However, a dose of 2 rals of the vaccine described in Example VI has generally been found to be effective in pigs up to 30 kg body weight. For pigs with a body weight above 30 kg, a dose of k ml of the 5 vaccim is suitable.

The following examples demonstrate the benefits obtained by using the vaccine; of the present invention for vaccination against pleuropneumonia as compared to known vaccines containing other additives.

Example VII.

Pigs weighing approximately 25 kg received a subcutaneous injection of vaccines A to E described in more detail below. Vaccination was repeated 3 weeks later. The dose was 2 ml, 5 ml and 6 ml, respectively. 3 weeks after the second injection, the pigs were challenged with H. pleuropneumoniae - 10 bacteria (about 10 H. pleuropneumoniae bacteria per animal) and 3 pigs not receiving vaccination were also challenged and used as control animals. The control animals died and the vaccinated animals survived. After slaughter, the 20 animals were examined to determine whether the infection had resulted in changes in the lungs and / or changes that could have affected the value of the slaughtered animal as fit for human consumption.

The vaccines used in this test vaccination were the following: AH pleuropneumoniae cells were grown on agar plates and harvested with a PBS solution (composition see Table V) containing 0.01% w / v solution of "Thiomersal "contained. The cell suspension, containing about 10 cells per ml, was extracted at 5 ° C for 30 minutes and then centrifuged, and 1 part by volume of Freund's incomplete additive was added to 3 vol. parts of the above layer. Dosage 2 ml (twice).

B. Like vaccine A, except that the extracted cells were boiled at 100 ° C for 2 hours before centrifugation. Dosage 2 ml (twice-35 times).

C. The cells were grown as vaccine A, harvested with PBS solution containing 0.01 w / v% solution of "Thiomersal" and adjusted to a density of 20 I.O.U. and then 50% Freund's incomplete additive was added. Dosage 5 ml (twice * f0 times) containing approximately 10 ^ H. pleuropneumoniae cells per ml - 8020132-29.

D. H. pleuropneuraoniae cells were grown in a flask to the density of 5 I.O.U. killed with formaldehyde and precipitated by the addition of Alhydrogel as described in Example IV. The precipitate was diluted with PBS solution to a concentration of 1 mg / ml at a level of about 10 ^ H. pleuropneumoniae cells per ml. Dosage 5 ml (twice).

E. A vaccine of the present invention having the composition given in Example VI and prepared essentially as described in Examples II, IV and V. Dosage 6 ml (twice).

The vaccination results are summarized in Table IX below.

Table IX.

Number of Clinical Changes Used Comments H. p.

vaccine * 'test symptoms of lungs in the slaughter animals taught A 10 2 / ΐ0 & ^ 0/109 ^ 1 / 10a ^ 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 20 E 6 0/6 0/6 0/6 N.D.

none 3 3/3 °) 3/3 - 3 / 3- (control) a) number of animals with symptoms (changes, comments) / number of animals.

B) Not determined.

c) 1 animal killed, 2 killed at death.

Table IX shows that vaccine E of the present invention is superior to known vaccines A to D in providing protection against infections caused by H. pleuropnea moniae bacteria. Vaccine E is the only vaccine * - in which neither symptoms nor changes of lungs and / or remarks during slaughter have been observed. Lung changes, i.e. scarring of the lung tissue as a result of a pleuropneumonia attack, were observed in 8 of 45 vaccinated animals. Chronic pleurisy was observed in 4 of the animals at slaughter, however, all 45 animals were acceptable for human consumption. Example VIII.

A further test vaccination was performed in 46 pigs and 2 animals were used for control purposes. The animals weighed about 25 kg. The vaccinations and challenge infections were performed in the manner described in Example VII. The dosages used for vaccination 8020152-30 were 2 times 2 ml.

The primary purpose of this test is to compare the effect of the Bordetella pertussis additive of the present invention on the effect of various other additives, some of which have already been used as such additives in the prior art.

The control animals survived to slaughter, however two of the vaccinated animals died before slaughter. One group L pig was sacrificed and clearly showed signs of pleural opioid mononies. One group E pig died of fighting, that is, for a reason unrelated to the present experiment. Examination of the lungs showed no evidence of pleuropneumonia. The slaughtered animals were examined for the purposes described in Example VII.

The compositions of the test vaccines are given below.

It should be noted that "vaccine" L is not a vaccine in the usual sense, however, the results obtained show that pleuropneumonia was not successfully controlled by pernasal and peroral administration of the active ingredient in the vaccine of the Present invention.

The vaccines used were the following: C, D, E. As in Example VII.

F. A vaccine of the composition of Example VI (Vaccine-E), except for a level of "Quil A" as an additive instead of Vaccinum pertussis. "Quil A" is a saponin derivative of known composition, described by K. Dalsgaard in Acta Vet. Scand., Suppl. 69 0978).

G. As F, except for a content of "Bortavac" as the additive in place of "Quil A", "Bortavac" is a Brondella bronchiseptica vaccine, which is marketed by the manufacturer Kitasato, Tokyo, Japan.

H. As F, except for a content of "Levoripercol" as the additive in place of "Quil A". "Levoripercol" is Levamisolum NFN anthelmintic, which is marketed by Lundbeck, 35 Copenhagen, Denmark. Levamiso lum is a non-proprietary name approved by NFN (The Nordic Pharmacopoeia Council).

I. As Vaccini E, except for a "Lymphocyte Proliferative Factor" (LPF) content as the additive in place of Vaccinum pertussis. The LPF was extracted from 8020132 -31-

Bordetella pertussis bacteria as described by S.I. Morse and J.H. Morse, see J. Exp. Med. 143 (1976), 1483-1502.

K. As Vaccim E, except for a content of Vacci number pertussis in an amount of 180 I.O.U.

5 L. H. pleuropneumoniae cells were grown on an agar plate, harvested and transferred to an aerosol, which was nebulized in the nose and mouth of the animals.

Of the aforementioned vaccines, 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.

Number of Clinical Changes Used Comments H. p.

vaccine · lung symptoms in the slaughtered animals learned C 3 0 / 3a ^ 0 / 3a) 0 / 3a ^ N.D.b ^ D 5 0/5 0/5 0/5 N.D.

E 10 0/10 0/10 1/9 ° ^ N.D.

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

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

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

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

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

L 2 2/2 2/2 1 / 1C) 1/2 25 none 2 2/2 2/2 2/2 N.D.

(control) a) Number of animals with symptoms (changes, comments) / number of animals.

b) Not determined.

30 c) 1 Animal died before slaughter.

Table X shows that clinical symptoms only occur in aerosol-treated animals and in the control.

Of the remaining 44 animals, only 2 showed changes in the lungs and only 3 gave rise to comments on slaughter, although serological studies showed that all animals had been affected by H. pleuropneumoniae.

Therefore, Table X shows that vaccination with an H. pleuro-pneumoniae vaccine, which contains killed H. pleuropneumoniae bacteria prepared according to the guidelines described in Examples II, IV and V above as the active ingredient, is very is effective in protecting pigs from pleuropneumonia attack. Although the 8020132 different additives tested may seem equally suitable according to Table X, the B. pertussis vaccine additive (or biomass and / or extracts thereof) used according to the invention is better and thus preferred, because it results in optimal protection and no or only a minor and short reaction at the injection site, while the known additives tend to give rise to granulomas at the injection site,

It will be understood that the foregoing description and examples are to be considered only as embodiments of the invention and it will be obvious to those skilled in the art that modifications and variations can be made therein without departing from the scope of the invention, such as defined in the appended claims. In particular, it will be appreciated that the invention is not limited to the use of the specific strain and / or subculture of H. pleuropneumoniae. Therefore, cultures obtained from this strain and / or subculture by natural and / or artificial selection and / or mutation, as well as cultures of each of the different serotypes are also within the scope of the present invention.

8020132

Claims (33)

1. Vaccine for the control of the infectious disease pleopneumonia in pigs caused by the micro-organism Haemophilus pleuropneumoniae, which extracts H. pleuropneumoniae-5 cells, parts of such cells and / or metabolism products thereof. contains the active ingredient and contains additives and a buffer, characterized by a content of a Bordetella pertussis vaccine, a B. pertussis biomass and / or extracts thereof as an additive. Vaccine according to claim 1, characterized by a content of Vaccinum pertussis prepared, identified and tested according to the European Pharmacopoeia, part III (1975) ^09, as an additive. Vaccine according to claim 1 or claim 2, characterized by a content of Vaccinum pertussis of 16 I.O.U. (international opacity units). k. Vaccine according to claims 1 to 3, characterized 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. Vaccine according to any of claims 1 to * f, characterized by a content of about 10 cells H. pleuropneumoniae per ml. 6. Vaccine according to any of claims 1 to 5, characterized by the following composition: Q H. pleuropneumoniae bacteria 10 cells Alhydrogel 0.03 ml "Thiomersaln 0.1 mg Vaccinum pertussis, Ph. Eur (160 IOU) 0.1 ml of sodium chloride phosphate buffer (pH 7) to 1 ml. 7. Process for the aerobic fermentation of the microorganism Haemophilus pleuropneumoniae in a liquid fermentation medium, which contains assimilable sources of carbon and nitrogen and suitable salts and other nutritional components commonly used in the fermentation of microorganisms, in particular Haemophilus species characterized by fermentation in an environment containing casamino acid, yeast extract, glucose and β-nicotineamide adenine dinucleotide (NAD) as essential ingredients. Process according to claim 7, characterized by * f0 fermenting in a liquid medium with the composition: f020132 ► -34- casamino acid 18.0 g yeast extract 14.0 g glucose 11.0 g NaCl 2.5 g 5 ΚΗ2Ρ0 ^ 0, 5 g msgi2.6h2o 0.1 g CaCl2 (1 w / v solution) 1.0 ml FeS0 ^ 7H20 (0.5 w / v solution, freshly prepared) 2.0 ml CuS0 ^. 5H20 (0.05 w / v solution) 1.0 ml 10 cystexne HCl (1 w / v solution) 2.5 ml NAD (2.5 w / v solution) 1, 0 ml of ion-exchanged water to 1000 ml. 9. A method according to claim 7 or claim 8, characterized by fermenting with stirring the fermentation medium during the fermentation. Process according to any of claims 7 to 9, characterized in that a substantially constant pH of the fermentation medium is maintained during the fermentation. A process according to any of claims 7 to 10, g -20 characterized by maintaining a pH from about 6.0 to about 7.9 in the fermentation medium. The method of claim 10 or claim 11, characterized by maintaining a pH from about 6.9 to about 7.5 in the ferentation medium. A method according to any of claims 10 to 12, characterized by maintaining a pH from about 7.1 to about 7.4 in the fermentation medium. . 14. Process according to any of claims 10 to 13, characterized by maintaining a pH of about 7.3 in the fermentation medium. A method according to any one of claims 7 to 14, characterized by fermenting in an environment with a substantially neutral initial pH. 16. A method according to claim 15, characterized by fermenting in an environment with an initial pH of about 7.2. A method according to any of claims 7 to 16, characterized by fermenting in an environment with an oxygen concentration of about 1 to about 30 ppm during the fermentation. A method according to claim 17, characterized by 8020132 -35- fermenting at an oxygen concentration of about 8 to about 20 ppm. 19. A method according to claim 17 or claim 18, characterized by fermenting at an oxygen concentration of about 8 to about 12 ppm. 20. The method of any one of claims 17 to 19, characterized by fermenting at an oxygen concentration of about 10 to about 12 ppm. A method according to any one of claims 17 to 20, g e -10 characterized by maintaining the desired oxygen concentration by varying the stirring speed and / or the air flow rate. The method of claim 21, characterized by fermenting at a stirring rate of about 12 to about 15 600 rpm. A method according to claim 21 or claim 22, characterized by fermenting at a stirring speed of about 500 rpm. 24. The method of claim 21, characterized by fermenting at aerobic conditions maintained by applying an air flow rate of about 0.5 liters of air per minute per liter of fermentation medium. A method according to claim 24, characterized by fermenting at an air flow rate of about 0.15 to about 0.4 liters of air per minute per liter of fermentation medium. A method according to claim 24 or claim 25, characterized by fermenting at an air flow rate of about 0.2 to about 0.3 liters of air per minute per liter of fermentation medium. A method according to any of claims 7 to 26, characterized by fermenting at a temperature of about 37 ° C. A method according to any of claims 7 to 27, characterized by fermenting a subculture of the strain H. pleuropneumoniae ATCC 27089 (NCTC 10976). The method according to claim 28, characterized by fermenting the subculture 4226 (State Veterinary Serum Laboratory, Copenhagen, Denmark) of the strain H. pleuropneumoniae. 30. Substrate for cultivation of microorganisms, particularly suitable for cultivation of the microorganism Haemophilus 8020132 36 -36- pleuropneumoniae, which are assimilable sources of carbon and nitrogen and suitable salts and other nutrients commonly used in the cultivation of micro-organisms, in particular Haemophilus species, characterized by a content of casamino acid, yeast extract, glucose and β-nicotine-amide-ade-nine-dinucleotine (NAD) as essential components. 31. Substrate according to claim 30, particularly suitable for the aerobic fermentation of H. pleuropneumoniae in a liquid fermentation medium, characterized by the following composition: casamino acid 18.0 g yeast extract 1.0 g glucose 11.0 g NaCl 2.3 g 15 ΚΗ2Ρ0 ^ 0.5 g MgCl2.6H20 0.1 g CaCl2 (1 w / v% solution) 1.0 ml FeS0 ^ .7H20 (0.5 w / v solution, fresh prepared) 2.0 ml CuSO5 .5H2 O (0.05 w / v solution) 1.0 ml 20 cysteine HCl (1 w / v solution) 2.5 ml NAD (2.5 wt ./vol.$ solution) 1.0 ml ion-exchanged water to 1000 ml. Substrate according to claim 30 or claim 31, characterized in that the substrate is adjusted to a pH of about 7.2. Substrate according to claim 30, particularly suitable for growing H. pleuropneumoniae on a solid substrate, such as on agar plates, characterized by the composition: casamino acid 6.0 g 30 yeast extract 25.0 g glucose 2.0 g NaCl 2 .5 g κη2ρο ^ 0.5 g MgCl2.6H20 0.1 g CaCl 2 (1 w / v% solution) 1.0 ml FeSO 4 .7H 2 O (0.5 w / v solution, freshly prepared) 0.5 ml cysteine * HCL (1 w / v. solution) 0.8 ml agar agar 16.0 g NAD (2.5 w / v solution) 1.0 ml * 10 ion exchanged water to 1000 ml. Substrate according to claim J, characterized in that the substrate is adjusted to a pH of approximately 7i2. ****** »020132