BE882619A - VACCINE FOR THE CONTROL OF PLEUROPNEUMONIA IN PIGS - Google Patents

Description

       

  At the beginning of 1960 bacteria of the genus Haemophilus were isolated in Great Britain, California and Argentina in herds of pigs which were attacked by pleuropneumonia. It was there

  
  <EMI ID = 1.1>

  
pneumoniae like the Argentine strain was named - which was the cause of the disease, pleuropneumonia. Since then attacks of the disease have been reported in

  
many countries, including Denmark, cf. P. Nielsen, North.

  
Vet. Med. 22 (1970), 240-245.

  
The disease has a very acute course and is often accompanied by high mortality and, as it occurs quite frequently in many herds of pigs, efforts have been made to try to develop a vaccine which could protect pigs against the disease . A common feature of these vaccines is their use of killed cells

  
of H. pleuropneumoniaé and, in most cases also, an addition of an adjuvant.

  
Vaccination tests were also carried out under field conditions, cf. R. Nielsen, North. Vet. Med. 28 (1976), 337-348. In these tests, agar plate cultures aged 6 or 24 hours of H. pleuropneumoniae are used as antigen. The cells are killed with formaldehyde and the vaccine contains 1010 cells per ml in a phosphate buffered saline (physiological) (abbreviated below as PBS) containing 0.2% formaldehyde and with the addition of an adjuvant. The vaccine is administered as 2 x 4 ml or 2 x 2 ml subcutaneous injections at 14 day intervals.

  
The first vaccination is carried out when the pigs are 9 weeks old. 2 weeks after the second vac-

  
  <EMI ID = 2.1> pleuropneumoniae. As adjuvants, 15% Alhydrogel (aluminum hydroxide gel) and incomplete Freund's adjuvant were used, cf. J. Freund, Am. Rev. Microbiol. 1 (1947), 291.

  
The results of these vaccination tests are summarized in Table I below.

TABLE I

  

  <EMI ID = 3.1>


  
It will be added that the use of Freund's incomplete adjuvant gives rise to the formation of granulomas at the site of the injection.

  
Attempts have also been made to obtain protection using 20 hour cultures killed with formalin as antigen and respectively Alhydrogel and incomplete Freund's adjuvant as an adjuvant. The dose injected is 2 times 5 ml containing 10 bacteria / ml. The first injection is given intramuscularly in pigs aged 50 days and the second injection 1 week later. After 1 week the pigs are infected. The result is significant evidence that the vaccine has a protective effect, but there is no significant indication that one adjuvant offers advantages over the other.

  
E. Scholl et al. , cf. Proc. Int. Pig Vet. Soc. Congress, Ames, Iowa, USA (1976) used killed cultures of H. pleuropneumoniae as antigens and vaccinated by two methods: 1) with increasing doses, namely, 0.5, 1 and 3 ml and 2) with 2 times 5 ml. All injections are given at an interval of 2 weeks. Serological test of blood samples shows that repeated injections of small doses lead to antibodies

  
in the blood, but not to sensitized lymphocytes, while high dose injections lead to sensitized lymphocytes but not to antibodies in the blood.

The same authors, cf. Proc. Int. Pig. Vet.

  
Soc. Congress, Zagreb, Yugoslavia (1978) used cultures of H. pleuropneumoniae killed with formalin to

  
concentrations of 10 9 bacteria / ml. Vaccination doses and vaccination methods are the same

  
than in the works cited above; but, in these latter works, a group of placebo pigs is included and the animals are placed in a herd of chronically infected pigs. Clinical symptoms were observed and, following slaughter, the lungs were examined and

  
the chest cavity. The results show a good &#65533; ffet

  
of vaccination, but no differences between the two vaccination methods.

  
M.F. de Jong, cf. Proc. Int. Pig Vet. Soc. Congress, Zagreb, Yugoslavia (1978), carried out vaccination tests with cultures of H. pleuropneumoniae

  
24 hours to which an incomplete adjuvant has been added

  
based on mineral oil. For each dose we use 20 mg
(wet weight) of antigen and two injections are made

  
at an interval of 3 weeks, the first vaccination when pigs are 10 weeks old. 3 weeks after the second vaccination, the pigs are challenged with 16 or 24 hour cultures. With 24 hour challenge cultures, pigs get sick, but recover after 1 week. On the other hand, pigs challenged with a 16-hour provocative culture die in the

  
36 hours. Provocation, a thing well known to man

  
of art, involves an experimental infection of animals tested with living cells of the infectious microorganism.

  
M.F. de Jong, loc. cit., then used 6 hour cultures for both vaccination and challenge. Pigs are provoked with different quantities of cells and the death rate is a function

  
the number of cells used. In addition, pigs which

  
have been vaccinated with 6 hour cultures are challenged with 16 hour cultures and 50% of pigs survive.

  
1 week later the surviving pigs are infected with a 6 hour culture and then they do not even show any more symptoms of disease.

  
The work on the vaccination tests mentioned above clearly shows the need to add an adjuvant to a vaccine containing antigens

  
of H. pleuropneumoniae in order to obtain a satisfactory effect from vaccination. The substances most commonly used as adjuvants are Alhydrogel and the adjuvant

  
Freund (complete or incomplete). Among these substances, Freund's adjuvant has so far been the most effective, but, as also pointed out, cf. R. Nielsen, loc. cit. (1976), this results in the formation of granulomas at the injection site and

  
this is why it is considered improper. Therefore the problem of providing an adjuvant which is effective but which does not interfere with animals has not yet been resolved.

  
by the experimental vaccinations previously reported.

  
Another unresolved problem is the preparation of an antigen for the production of the vaccine. This issue

  
lies in the fact that H. pleuropneumoniae has quite poor growth on the conventional substrates used previously, which imposes very restrictive limits as to the quantity of vaccine that it is possible to produce.

  
Thus J. Nicolet, cf. Zbl. Bakt. I Abt. Orig.
216 (1971), 487-495, in its serological studies used a substrate comprising PPLO-agar (cf. DIFCO-Manual IX,

  
  <EMI ID = 4.1>

  
pneumoniae) to which were added 0.1% glucose, 2.5% yeast extract, 5% horse serum and 5 mg / liter

  
NAD (beta-nicotinamide-adenine-dinucleotide). The same substrate is used by R. Nielse, loc. cit. (1976).

  
M.F. de Jong, loc. cit., used a brain-heart infusion containing NAD as a substrate and M. Kilian et al., cf. Int. J. Syst. Bacteriol. 28 (1978), 20-26,

  
have carried out taxonomic studies of H. pleuropneumoniae, cultivating the strains on a substrate comprising:

  
1% neutralized bacteriological peptone ("Oxoid") 1% yeast extract,

  
1% glucose,

  
10 mg / liter of NAD and

  
saline as disclosed by L.V. Holdeman and W.E.C. Moore in Anaerobe Laboratory Manual,

  
3rd ed. , Virginia Polytechnic Institute, Anaerobe Laboratory, Blacksburg (1975).

  
Some other authors have cultivated H. pleuropneumoniae in liquid cultures, but with rather poor results.

  
An object of the invention is to provide a new and improved vaccine for the effective control of pleuropneumonia in pigs, by also providing a new and improved adjuvant for this vaccine, without the side effects associated with adjuvants of the prior art .

  
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, so that it is possible to produce an effective vaccine by suf- quantities. fisantes for an extended vaccination if the thing is desired.

  
Another 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 cultivated on substrates of the prior art .

  
Yet another object of the invention is to provide a method for combating pleuropneumonia in pigs by administering the vaccine of the present invention.

  
These and other objectives are achieved by providing a vaccine which comprises Haemophilus pleuropneumoniae cells, parts of these cells, extracts and / or metabolic products thereof, which are obtained by culture of a strain of H. pleuropneumoniae

  
in a liquid culture based on the substrate disclosed by

  
S.M. Cohen and M.W. Wheeler in Am. J. Publ. Hlth. 36 (1946),
371-376, this vaccine also comprising, as an adjuvant, aluminum hydroxide gel and / or incomplete or complete Freund's adjuvant and / or Bordetella pertussis vaccine, a biomass and / or extracts thereof , as well as a sterile buffer solution as a diluent.

  
The bacterium used for the production of the vaccine of the present invention is a strain of Haemophilus pleuropneumoniae, a known microorganism deposited in various collections of cultures, see below, and available from

  
of these.

  
Investigations have shown that there are at least 6 different serotypes of H. pleuropneuinoniae, cf.

  
M. Kilian et al. , loc. cit. (1978). This reference from

  
the literature also explains why the name of H. pleuropneumoniae is considered to be the correct name of the organism causing pleuropneumonia in pigs and it

  
gives a general overview of the characteristics of the microorganism.

  
Among the various serotypes of H. pleuropneumoniae described in the above reference of the literature, some have been deposited, thus serotype 1 in the American Type Collection, RocKville, Md., U.S.A. under the N [deg.] ATCC

  
27088, serotype 2 under the. N [deg.] ATCC 27089 and serotype 3 under N [deg.] ATCC 27090. In addition serotype 2 has been deposited

  
in the National Collection of Type Cultures, Colindale, London, United Kingdom under N [deg.] NCTC 10976 and these three serotypes have all been deposited in the Czechoslovak Collection of Microorganisms, Brno, Czechoslovakia, under

  
N [deg.] CCM 5869, CCM 5870 and CCM 5871, respectively.

  
In addition, M. Kilian et al. loc. cit. (1978) describe serotypes designated by serotypes 4, 5 and NT. However, deposit numbers are not indicated.

  
Applicant's own investigations

  
confirmed that the bacteria of H. pleuropneumoniae

  
are small, stationary gram-negative sticks that can take a number of configurations depending on the growing medium and surrounding conditions.

  
Thus, on agar plates they are most often small sticks of the shell or short kind, while

  
that in liquid cultures they can vary from rods in the form of shells to slender sticks longer or thick, with the exception (quite rare) even of pleomorphic filamentary configurations.

  
Grown on agar plates on the substrate

  
of the present invention (for the composition refer

  
in Table III below), the bacteria of H. pleuropneumoniae have the configuration of small shell-like sticks

  
and the individual colonies have a diameter of up to 3 mm after 48 hours at 37 [deg.] C. Fermented in the nutritive broth of the present invention (for the composition refer to Table IV below), the bacteria of H. pleuropneumoniae are short sticks which often are in shorter or longer chains.

  
At the biochemical level, the bacteria of H. pleuropneumohiae behave as described by M. Kilian, J. Gen. Microbiol. 93 (1976), 9-62, having the following characteristic properties which together make them different from other Haemophilus species: dependence on factor V, ability to synthesize porphyrin from delta-aminolevulinic acid, production urease and ability to ferment mannitol, xylose and deoxyribose.

More specifically, a strain of

  
H. pleuropneumoniae which was isolated from a pig suffering from pleuropneumonia. This strain is of serotype 2,

  
cf. R. Nielsen, loc. cit. (1976) and was submitted to the Statens Veterinaere Serumlaboratorium (State Veterinary Serum Laboratory), Copenhagen, Denmark, where it was designated as subculture 4226.

  
The substrate used in the implementation of

  
the present invention is based on the substrate disclosed by

  
S.M. Cohen and M.W. Wheeler, loc. cit., having the composition given in Table II below.

TABLE II

  

  <EMI ID = 5.1>


  
  <EMI ID = 6.1>

  
For the purposes of the present invention, this substrate is modified so as to improve the growth of H. pleuropneumoniae and to provide a suitable substrate for the preparation of an H. pleuropneumoniae vaccine.

  
  <EMI ID = 7.1>

  
if desired. Two different modifications are used for culture on agar plates and respectively fermentation in a liquid medium.

  
For agar plates, a substrate comprising casamino acid is used as essential ingredients,

  
  <EMI ID = 8.1>

  
adenine dinucleotide (hereinafter called NAD). Furthermore,

  
this substrate must contain suitable salts and other nutritive ingredients conventionally used in the culture of microorganisms, in particular of the Haemophilus species. A substrate especially preferred to the composition given in Table III below.

TABLE III

  
  <EMI ID = 9.1>

  

  <EMI ID = 10.1>


  
This substrate is prepared in the following manner:
casamino acid, yeast extract, glucose, NaCl,

  
  <EMI ID = 11.1>

  
cysteine.HCl and agar-agar and regulates the volume to 1000 ml by the addition of H2O treated by ion exchange. The solution is heated to the boil (in order to dissolve the agar) and it is autoclave at 121 [deg.] C under a pressure of 2 atmospheres

  
  <EMI ID = 12.1>

  
the NAD and the substrate is poured into petri dishes.

  
The substrate of the present invention for use in fermentation in a liquid medium also includes casamino acid, yeast extract, glucose and NAD as essential ingredients. This substrate must contain suitable salts and other nutritive ingredients commonly used in the fermentation of microorganisms, in particular of the Haemophilus species. A substrate especially preferred to the composition given in Table IV below.

TABLE IV

  
Substrate for fermentation in liquid medium

  

  <EMI ID = 13.1>


  
This substrate, abbreviated as CAY substrate, is prepared as follows:

  
casamino acid, yeast extract, glucose,

  
  <EMI ID = 14.1>

  
CuS04.5H20 and cysteine.HCl, the volume is adjusted to 1000 ml by the addition of H2O treated by ion exchange and the solution is autoclave at 121 [deg.] C under a pressure of 2 atmospheres for 15 minutes. After cooling, the NAD is added and the substrate is ready for use.

  
The two substrates disclosed in Tables III and IV include assimilable sources of carbon and nitrogen which can be assimilated by the microorganism H. pleuropneumoniae. On both substrates we obtain an abundant and dense growth of the organism.

  
The fermentation process is as follows:

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

  
Method A

  
The bacteria are cultivated at 37 [deg.] C for 18 hours on agar plates with a substrate having a composition like that indicated in Table III. The cells are harvested by the addition of 1 ml of PBS (composition given in table v below), with subsequent abrasion of the cells. The cells are transferred to ampoules which are immediately cooled to -25 [deg.] C., then they are transferred to a freeze dryer and are freeze dried.

TABLE V

  
Composition of PBS

  

  <EMI ID = 15.1>


  
The salts are dissolved in distilled water and autoclave at 12 l [deg.] C under a pressure of 2 atmospheres for 15 minutes. pH = 7.3.

  
Method B

  
This method is similar to Method A, but use 1 ml of skim milk for each plate

  
instead of PBS. The skimmed milk was treated hot at 100 [deg.] C for 15 minutes for the next 3 days.

  
Method C

  
The bacteria are cultivated for 6 hours in a bain-marie in CAY substrate (see Table IV). The fully grown suspension of bacteria is centrifuged and the bacterial cells are resuspended in sterile skim milk, which is transferred to vials which are cooled and lyophilized as described in Method A.

  
The bacteria are restored from the lyophilized vials by the addition of substrate followed by inoculation on agar plates and in tubes containing the liquid substrate.

  
Live cultures are maintained by daily inoculation on fresh agar plates containing the substrate of Table III. Plates are subject to

  
incubation in an incubator at 37 [deg.] C.

  
A preculture is prepared by propagation of the bacteria. This is done by inoculation from an agar plate in a vial containing 50 ml of liquid CAY substrate. The flask is subjected to incubation in an incubator at 37 [deg.] C

  
  <EMI ID = 16.1>

  
inoculation of the second preculture.

  
The second preculture consists of 500 ml of liquid CAY substrate in a 1 liter conical flask which is subjected to incubation on a water bath with continuous shaking. The temperature is 32 [deg.] C and the incubation period is 18 hours.

  
The second preculture is used for the main fermentation during 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, namely by dissolving the casamino acid, the extract

  
yeast, glucose, NaCl, KH2PO4 and MgCl2.

  
6H20 in ion exchange treated water, adjusting the pH to around 7.2, adding the remaining ingredients

  
with the exception of NAD, autoclaving the medium at a temperature of at least about 121 [deg.] C under a pressure of at least about 2 atmospheres for at least 15 minutes,

  
  <EMI ID = 17.1>

  
controlling and readjusting the pH to about 7.2, and adding the NAD.

  
Then the fermenter is inoculated with the second preculture of H. pleuropneumoniae obtained as described above. During fermentation, a temperature is preferably maintained of around 37 [deg.] C and air circulation is used through the medium in order to maintain an adequate concentration of oxygen in the medium. This can be done in part by controlling the air flow rate and in part

  
by adjusting the speed of the agitator device of the fermenter, thus taking into consideration that these parameters are mutually dependent, in the sense that an increase in

  
one of these leads to an increased concentration of oxygen which, in turn, may require a decrease in the other parameter.

  
For the fermentation of H. pleuropneumoniae, the oxygen concentration can vary from approximately 1 to approximately

  
30 ppm, the most suitable concentration being around

  
8 to about 20 ppm, in particular about 8 to about 12 ppm, more specifically still from about 10 to about 12 ppm.

  
The air flow and stirring speed required for

  
to maintain such an oxygen concentration will obviously depend on the dimensions, the equipment and the 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 from about 0.15 to about 0.4 liters, more specifically d 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 capacity of about 20 liters. For this fermenter an appropriate stirring speed is from about 200 to about 600 rpm, in particular from about 500 rpm.

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

  
During fermentation it is preferable

  
to maintain a substantially constant pH. A suitable pH is in the range of about 6.0 to about

  
7.9, in particular from approximately 6.9 to approximately 7.5, more specifically from approximately 7.1 to approximately 7.4, preferably approximately 7.3. This can be accomplished by the addition of a base, preferably an aqueous solution of sodium hydroxide if necessary.

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

  
The fermenter is preferably provided with

  
means allowing automatic addition of pH adjustment base and / or anti-foaming agent during fermentation. However the addition can also be done manually

  
if it's necessary.

  
During fermentation, samples are taken intermittently and used to determine cell density. The density is measured with a "Corning 252 colorimeter" at a wavelength of 600 nm and expressed as a number of colony-forming units (CFU) using a standard reference curve. The purpose of this density measurement

  
  <EMI ID = 18.1>

  
fermentation at the most appropriate time for the subsequent production of a vaccine, i.e. when the phase

  
in which the bacteria show exponential growth is 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 period, for example overnight, the suspension is subjected to cantrifugation, which gives a supernatant part containing formaldehyde and a precipitate comprising the cells of H. pleuropneumoniae and Alhydrogel. The precipitate is collected in a sterile container. A sample is taken to verify that the bacteria have been killed.

  
The following examples will 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 the present invention.

  
The fermenter is a "Biomat E 20" (Moeller

  
  <EMI ID = 19.1>

  
nir 20 liters of substrate in each fermentation and being equipped with a stirring means, means which allows manual variation of the stirring speed, an instrument which continuously indicates the stirring speed (in revolutions / minute) , means for supplying sterile air, means for continuous measurement and recording of the oxygen concentration of the fermentation medium, continuous means for automatic measurement and adjustment of the temperature at a predetermined value, of a means for continuous measurement and recording of the pH of the fermentation medium and for the manual or automatic addition of pH control bases or acids, of a means for controlling the foaming in the fermenter and for the automatic addition of an anti-foaming agent if necessary, and of a means for taking samples during fermentation

  
The fermenter is charged with 17 liters of water treated by ion exchange and one begins to agitate. Then add 306 g of casamino acid, 238 g of yeast extract,

  
187 g glucose, 42.5 g NaCl, 8.5 g KH2PO4 and 1.7 g

  
  <EMI ID = 20.1>

  
42.5 ml of a 1% w / v solution of cysteine.HCl. The fermenter is closed and autoclaved for 15 minutes at
121 [deg.] C under a pressure of 2 atmospheres then it is cooled to
36 [deg.] C. 17.0 ml of a 2.5% w / v solution of NAD is then added. This procedure leads to a fermentation medium having the composition given in Table IV, with a pH of 7.45.

  
The fermenter is inoculated with 600 ml of a second preculture of H. pleuropneumoniae prepared from a stock of lyophilized bacteria and cultured as described above. The fermentation period is 7 hours and 50 minutes and samples are taken intermittently and used to determine the absolute density of cells in the fermentation medium. The density is measured with the "Corning 252 Colorimeter" at a wavelength of 600 nm and is converted into cells per ml using a standard reference and a conversion table which makes it possible to plot a growth curve and determine when the exponential growth phase of bacteria has ended.

  
This fermentation is carried out mainly as experimental fermentation and that is why

  
we have taken the liberty to proceed in a somewhat "normal" manner, which includes the fact that the pH is not adjusted during fermentation and that no anti-foaming agent is added.

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

  
It will be seen that the oxygen concentration, which should preferably be 8 to 10 ppm, decreases drastically during the fermentation, in particular in the exponential growth phase. This is combated by partly increasing the air flow and partly the stirring speed. We will also see that the pH decreases to a final pH of 5.30.

  

  <EMI ID = 21.1>


  

  <EMI ID = 22.1>
 

  

  <EMI ID = 23.1>


  

  <EMI ID = 24.1>
 

  
The results from Table VI were used to plot the growth curve shown in Figure

  
1 of the drawings. In the figure, the abscissa indicates the time after inoculation and the ordinate indicates the number of cells per ml of fermentation medium. Note that the ordinates are represented with a logarithmic scale. The figure shows that the exponential growth phase ends about 5 hours after inoculation, which means that the fermentation should have been stopped

  
at this point if the medium was intended to be used for the production of a vaccine. However, as this fermentation is an experimental fermentation, it is continued until the stationary growth phase.

  
Fig. 1 of the drawings also allows to calculate

  
the time (Tg) necessary to double the bacteria content in the fermentation medium during the exponential growth phase. Tg is given by the formula:

  

  <EMI ID = 25.1>


  
  <EMI ID = 26.1>

  

  <EMI ID = 27.1>


  
where C2 is the number of cells per unit volume at time

  
  <EMI ID = 28.1>

  
In Figure 1 the exponential growth phase is represented by the rectilinear part of the curve. We see that this phase extends from no later than 1 hour until about 5 hours after inoculation. By using this part of the curve (results marked by a circle in FIG. 1, namely the samples bearing? 6 to 18) for a calculation according to formula (II), we see that:

  

  <EMI ID = 29.1>


  
  <EMI ID = 30.1>

  
According to formula (I) this means that:

  

  <EMI ID = 31.1>


  
which equals 39.6 minutes.

Example 2

  
This example describes a "normal" fermentation

  
  <EMI ID = 32.1>

  
adjusted to around 7.2 an anti-foaming agent is automatically added if necessary, more vigorous stirring is maintained and the oxygen concentration

  
is continuously maintained at a relatively high level.

  
The fermenter is identical to the fermenter used in Example 1. The volume of the fermentation medium is approximately 20 liters of which the inoculation medium provides 0.5 liters, the balance being 19 liters of water treated by exchange of ions containing casamino acid, yeast extract, glucose and salts in amounts producing

  
a CAY substrate whose relative composition is given in Table IV. Loading the fermenter, autoclaving,

  
the cooling and the subsequent addition of NAD are carried out as described in Example 1. The initial pH of the medium is

  
  <EMI ID = 33.1>

  
When the inoculation medium has been added, fermentation is started and continued for 7 hours

  
and 30 minutes. The inoculation medium consists of

  
500 ml of a second preculture of H. pleuropneumoniae,

  
ATCC 27089 strain, subculture 4226, grown from

  
as described above.

  
During fermentation the pH is maintained at

  
an almost constant value of approximately 7.2 by automatic addition of a 1 N NaOH solution when necessary. Stirring is carried out at 500 revolutions / minute and the oxygen concentration is maintained between approximately 8 and approximately 12 ppm. This is achieved by increasing the air flow when necessary. A commercial brand anti-foam agent "Silicone RS Antifoam C. DAK" is automatically added when necessary.

  
Samples are taken during fermentation and analyzed as described in Example 1 to be able to follow the growth rate. The fermentation period is 7 hours and 30 minutes.

  
The fermentation results are summarized in Table VII.

  

  <EMI ID = 34.1>


  

  <EMI ID = 35.1>
 

  

  <EMI ID = 36.1>


  

  <EMI ID = 37.1>
 

  
The results given in Table VII were used to plot the curve shown in Figure 2 of the drawings. The abscissa and the ordinate have the meanings given for figure 1, cf. Example 1. From FIG. 2, it can be seen that the fermentation is stopped while the microorganism is still in the exponential growth phase, although the growth rate is slightly less pronounced than in the initial phase of the fermentation. , a slight decrease in the slope of the curve occurring at around 3 hours and 30 minutes

  
after inoculation.

  
Measurements indicated with circles

  
in Figure 2 were used for calculations according to formulas (I) and (II) of Example 1, partly for the initial phase (phase 1, samples

  
  <EMI ID = 38.1>

  
samples? 15 to 27) of fermentation. The units used in the calculations in Example 1 are also used in these calculations, which gives the following results.

  

  <EMI ID = 39.1>


  
which equals 45.5 minutes
  <EMI ID = 40.1>
 
  <EMI ID = 41.1>
 which equals 75.7 minutes.

  
A comparison of the results obtained according to Examples 1 and 2 shows that although the process of iron-

  
  <EMI ID = 42.1>

  
the lowest, which means faster growth

  
bacteria in the fermenter and therefore a shorter fermentation period before the broth is ready for use in the production of a H. pleuropneumoniae vaccine, it also leads to a faster transition to the stationary growth phase , namely after a fermentation period of around 5 hours, while "normal" fermentation leads to an extended period

  
  <EMI ID = 43.1>

  
more than 7 1/2 hours. Most important . "normal" fermentation also leads to a higher yield of cells per unit volume when the broth is already

  
  <EMI ID = 44.1>

  
cells per ml, compare with approximately 27 x 108 cells per ml in Example 1 (sample? 18 where the exponential growth phase ends). Therefore, from a commercial point of view (vaccine production) the process

  
"Normal" fermentation of Example 2 is preferred because the greater consumption of time is more than offset by the higher yield of cells usable in subsequent vaccine production.

Example 3

  
This example is a comparative example which shows

  
that the fermentation of H. pleuropneumoniae, subculture

  
4226, on a known substrate, leads to a much slower growth rate and a much lower yield than the fermentation in the substrate of the present invention.

  
The fermenter is identical to that used in Examples 1 and 2.

  
  <EMI ID = 45.1>

  
crobiology "published by E. Merck, Darmstadt, Federal Republic of Germany, to which was added 6 mg of NAD per liter of broth. The fermenter is loaded with 17 li-

  
  <EMI ID = 46.1>

  
is 7.3.

  
As a inoculation medium, a second preculture of H. pleuropneumoniae ATCC 27089, subculture is added.
4226. This preculture was cultivated as described above. During fermentation we maintain a

  
  <EMI ID = 47.1>

  
let it decrease to a final pH of 6.85. The initial oxygen concentration is 10 ppm, but it drops very quickly to 0, partly because the air flow is low and partly because the agitation is immediately stopped after inoculation and not resumed until what a

  
  <EMI ID = 48.1>

  
Therefore, it is a characteristic of this fermentation that it must take place under almost anaerobic conditions to obtain an acceptable yield which, nevertheless, is low, cf. table VIII below.

  
The fermentation period is 6 hours and

  
  <EMI ID = 49.1>

  
sampled and analyzed in the manner and for the parameter described in Example 1. The results of the fermentation are summarized in Table VIII.

  

  <EMI ID = 50.1>


  

  <EMI ID = 51.1>
 

  

  <EMI ID = 52.1>


  

  <EMI ID = 53.1>
 

  
The results from Table VIII were used to plot the growth curve shown in Figure 3 of the drawings. The abscissas and the ordinates represent the same units as those cited in Example 1 for FIG. 1. By comparing the curves respectively in FIGS. 1 and 2, and in FIG. 3, it is clear that the growth rate of H. pleuropneumoniae is substantially faster in the CAY substrate of the present invention (fig. 1 and 2) than in the known CASO broth
(Figure 3), this being confirmed by calculations in accordance <EMI ID = 54.1>

  
under.

  
FIG. 3 shows that the exponential growth phase of the bacteria ends approximately 5 hours after inoculation and that this phase, as was the case in the fermentation of Example 2, comprises two different phases, the phase of which 1 ends approximately 3 1/2 hours after inoculation, i.e. approximately when the resumption of agitation leads to a higher oxygen concentration.

  
  <EMI ID = 55.1>

  
are made as described in example 1. Again the base for the calculations is constituted by the points of the curve of figure 3 which are represented with circles. The results are as follows:

  

  <EMI ID = 56.1>
 

  

  <EMI ID = 57.1>


  
  <EMI ID = 58.1>

  
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 that of fermentation on CASO broth, namely 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 broth is even even slower than the growth rate in phase 2 of Example 2 on CAY substrate (Tg = 78 minutes versus 75.7 minutes).

  
  <EMI ID = 59.1>

  
surprisingly higher than the yield on CASO broth
(26.4 x 108 cells per ml in Example 1 at the end of the exponential growth phase and 37.2 x 108 cells

  
per ml in Example 2, against 10.2 x 108 cells per ml

  
in example 3). Consequently, the yields on the substrate of the present invention are 2.6 and 3.6 times, respectively, the yields on the known substrate.

Example 4

  
After a fermentation period of 7 1/2 hours, the fermentation described in Example 2 is stopped by the addition of 60 ml of concentrated formaldehyde solution and

  
600 ml of Alhydrogel. The added Alhydrogel is a 2% w / v solution of aluminum hydroxide. After vigorous stirring overnight (for about 18 hours), the fermentation broth is transferred to a storage tank from which it is pumped in a disc centrifuge and separated into an aqueous supernatant part of formaldehyde which is separated and separated. a precipitate comprising the cells of H. pleuropneumia and the Alhydrogel. The volume of precipitate represents approximately 2% of the initial volume of broth. The precipitate is collected in a container. Samples are taken and it is checked that all the bacteria have been killed.

  
The procedure described in this example

  
is performed under aseptic conditions, i.e. the added solutions and the equipment used have been sterilized before coming into contact with the fermentation broth.

Example 5

  
The precipitate collected as described in the example

  
4 is used for the production of an H. pleuropneumoniae vaccine. A vaccine is obtained by diluting the precipitate with

  
phosphate buffered saline (physiological), solution
(PBS), having the composition shown in Table V, up to about the desired density for the vaccine. Then we add

  
  <EMI ID = 60.1>

  
and optionally Alhydrogel, and the precipitate, PBS solution, "Thiomersal" and Alhydrogel are suspended in a sodium chloride-phosphate buffer having a pH of 7, stirred for 20 minutes and take a sample for analysis. Based on this analysis, the density of the vaccine is adjusted to the desired value. Then a suspension of Bcrdetella pertussis (vaccine of

  
  <EMI ID = 61.1>

  
regulate the density and pour the vaccine into capped vials.

  
It will be understood that all of the stages described in this example are carried out under aseptic conditions.

  
For subsequent use in vaccination of pigs, it has been found that a suitable density of the vaccine

  
is around 20 I.O.U. and therefore we prefer that

  
the precipitate of example 4 is diluted with the solution

  
of PBS 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 B. pertussis suspension. It has also been found that a suitable concentration of the "Thiomersal" adjuvant is about 0.01% w / v and that the Alhydrogel adjuvant should preferably be present at a concentration

  
  <EMI ID = 62.1>

  
Alhydrogel it must be taken into account that at least most of this adjuvant has already been added during the treatment of the fermentation broth described in Example 4. This generally assumes that a minor adjustment of the Alhydrogel content is only necessary and it may even be superfluous to add Aihydrogel at the same time as "Thiomersal".

  
The suspension of Bordetella pertussis which, if desired, can also be added in the form of biomass of B. pertussis and / or extracts thereof,

  
is the ingredient in the H. pleuropneumoniae vaccine of the present invention which leads to the surprisingly superior properties and activities of the vaccine of the invention when used for the vaccination of pigs against pleuro-

  
  <EMI ID = 63.1>

  
Neither the B. pertussis vaccine itself
(biomass and / or extracts), nor the method for its production do not form part of the present invention, which implies that any vaccine of this kind can be used as an adjuvant in the H. pleuropneumoniae vaccine of the invention, provided that the B. pertussis vaccine as well as the final vaccine of the present invention meet all the requirements imposed on the vaccines according to national and / or 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 the European Pharmacopoeia, Vol. III (1975), 409, which satisfies the tests mentioned here.

  
An appropriate amount of the adjuvant, the suspension of B. pertussis, in the H. pleuropneumoniae vaccine of the invention is about 16 I.O.U. vaccine, which can be adequately obtained by adding 100 ml of the above

  
  <EMI ID = 64.1>

  
1 liter of suspension obtained by mixing the precipitate

  
of Example 4, the PBS solution, the "Thiomersal", the Alhydrogel when used, and the sodium phosphate chloride buffer.

  
  <EMI ID = 65.1>

  
"Corning 252 colorimeter" at a wavelength of 600 nm as described above.

  
The vaccine is transferred to capped vials

  
  <EMI ID = 66.1>

Example 6

  
Following the procedure described in Example 5, an H. pleuropneumoniae vaccine is prepared comprising:

  

  <EMI ID = 67.1>


  
sodium chloride-phosphate buffer (pH 7) to make 1 ml. Clinical investigations

  
It has been found that the vaccine of the present invention is effective in combating pleuropneumonia in pigs exposed to infection by the microorganism Haemophilus pleuropneumoniae and that it is therefore indicated for the prophylactic treatment of pigs, in particular seronegative pigs, before they travel to an environment which may already be infected with H. pleuropneumoniae, or where the risk of such infection may exist. In

  
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, a subcutaneous injection being preferable.

  
To ensure effective vaccination, the vaccine of the present invention is administered twice, with an adequate interval between injections, which is preferably not less than 3 weeks. The dose administered will obviously depend on the body weight of the pigs, the potency of the vaccine, the risks of side effects and the nature of these effects when they exist, as well as other factors which are commonly taken into account when vaccination is carried out, regardless of the active substance in the vaccine. However, it has been found that a dose of 2 ml of the vaccine described in Example 6 is generally effective in pigs

  
  <EMI ID = 68.1>

  
weighing more than 30 kg, a 4 ml dose of the vaccine may be suitable.

  
The following examples show the advantages obtained by using the vaccine of the present invention for vaccination against pleuropneumonia, by making a

  
  <EMI ID = 69.1>

  
other adjuvants.

Example 7

  
45 pigs weighing about 25 kg are given a subcutaneous injection of the vaccines A to E described in more detail below. 3 weeks later the vaccination is repeated. The dosage is 2 ml, 5 ml and 6 ml respectively.

  
3 weeks after the second injection, the pigs are caused by H. pleuropneumoniae bacteria (approximately

  
1010 bacteria of H. pleuropneumoniae per animal), and 3 pigs which did not receive vaccination are also provoked and used as controls. Control animals die and vaccinated animals survive. After slaughter,

  
animals are examined to determine if the infection has resulted in changes to the lungs and / or changes that may affect the value of the animal slaughtered for delivery to human consumption.

  
The vaccines used in this vaccination test are as follows.

  
A. H. pleuropneumoniae cells are grown on agar plates and harvested with a solution of

  
PBS (for the composition see Table V) containing a 0.01% w / v solution of "Thiomersal". The suspension

  
  <EMI ID = 70.1>

  
fugée and add 1 part by volume of incomplete adjuvant

  
of Freund to 3 parts by volume of the supernatant part.

  
Dosage 2 ml (2 times).

  
B. Like vaccine A, except that it is boiled

  
  <EMI ID = 71.1>

  
C. The cells are cultured as for vaccine A, collected with a PBS solution containing a 0.01% w / v solution of "Thiomersal", adjusted

  
at a density of 20 I.O.U. and then 50% of incomplete Freund's adjuvant is added. Dosage 5 ml (2 times) containing approximately 10 9 cells of H. pleuropneumoniae per ml.

  
D. H. pleuropneumoniae cells are cultivated in a flask until the density reaches 5 IOU, they are killed with formaldehyde and precipitated by the addition of Alhydrogel as described in Example 4. diluted

  
precipitate with PBS solution to a concentration

  
  <EMI ID = 72.1>

  
E. Vaccine of the present invention of composition given in Example 6 and prepared substantially as described in Examples 2, 4 and 5. Dosage 6 ml (2 times).

  
The results of the vaccination are summarized in the table below.

TABLE IX

  

  <EMI ID = 73.1>


  
a) number of animals with symptoms (changes, remarks) / number of animals tested. b) not determined. c) 1 dead animal, 2 are killed while dying.

  
From Table IX, we see that the E vaccine

  
  <EMI ID = 74.1>

  
of the prior art in providing protection against

  
infections caused by H. pleuropneumoniae bacteria. The E. vaccine is the only vaccine for which no symptoms or changes in the lungs are observed and / or comments made at slaughter. Changes in the lungs,

  
namely scars in the lung tissue following an attack of pleuropneumonia, are observed in 8 of
45 vaccinated animals. At slaughter, chronic pleurisy was noted in 4 of the animals. Nevertheless, all 45 animals are acceptable for human consumption.

Example 8

  
Another vaccination test is carried out on 46 pigs and 2 animals are used as a control. The animals weigh approximately 25 kg. The vaccinations and infections caused are carried out as described in Example 7. The dosages used for the vaccination are 2 times 2 ml.

  
The main purpose of this test is to compare the effect of the adjuvant Bordetella pertussis of the present invention with that of various other adjuvants, some of which have already been used as adjuvants in the prior art.

  
Control animals survived to slaughter, but 2 of the vaccinated animals died before slaughter. A group L pig is killed; this clearly shows signs of pleuropneumonia. Group E pig is dead

  
following a battle, in other words for a reason unrelated to the present test. Examination of the lungs revealed no signs of pleuropneumonia. The slaughtered animals were examined for the purposes indicated in Example 7.

  
The compositions of the test vaccines are given below. Note that the "L vaccine" is not a vaccine in the common sense of this term; however, the results obtained demonstrate that pleuropneumonia is not successfully controlled by pernasal and peroral administration of the active ingredient in the vaccine of the present invention.

The vaccines used are:

  
C, D, E: as in Example 7.

  
F. vaccine of the composition of Example 6
(vaccine E), with the exception of a "Quil A" content as an adjuvant instead of Vaccinum pertussis. The "Quil A" is

  
a saponin derivative of known composition, which was disclosed by K. Dalsgaard in Acta Vet. Scand., Suppl. 69 (1978).

  
G. Like F, apart from a content of "Bortavac" as an adjuvant instead of "Quil A". "Bortavac" is a commercial Bordetella bronchiseptica vaccine sold by

  
the producer, Kitasato, Tokyo, Japan.

  
H. Like F, apart from a content of "Levoripercol" as an adjuvant instead of "Quil A". The "Levoripercol" is

  
Levamisolum NFN anthelminticum sold by the company Lundbeck, Copenhagen, Denmark. Levamisolum is an unprotected name approved by the NFN (The Nordic Pharmacopoeia Council).

  
  <EMI ID = 75.1>

  
"Proliferative Factor Lymphocyte" (LPF) as an adjuvant

  
instead of Vaccinum pertussis. The LPF is extracted from Bordetella pertussis bacteria as described by S.I. Morse and J.H.

  
  <EMI ID = 76.1>

  
K. Like vaccine E, apart from a content of Vaccinum pertussis in an amount of 180 I.O.U.

  
L. Cells of H. pleuropneumoniae are cultured on an agar plate, harvested and transferred to an aerosol, which is atomized in the nose and mouth of the animals.

  
Among the vaccines listed above,

  
  <EMI ID = 77.1>

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

PAINTINGS

  

  <EMI ID = 78.1>


  
) number of animals with symptoms (changes, re. marks) / number of test animals. b) not determined. c) 1 animal dies before slaughter.

  
From Table X we see that clinical symptoms appear only in animals treated with aerosol and in control animals. Only 44 restani animals 2 show changes in the lungs and

  
  <EMI ID = 79.1>

  
that it can be demonstrated by serological examinations that all animals have been attacked by H. pleuropneumoni:


    

Claims (34)

Table X therefore shows that vaccination with an H. pleuropneumoniae vaccine comprising killed H. pleuropneumoniae bacteria, prepared according to the instructions disclosed in Examples 2, 4 and 5 above as active ingredient, is very effective for the protection of pigs against an attack of pleuropneumonia. Although the various adjuvants tested may appear to be very suitable according to Table X, the B. pertussis vaccine adjuvant (its biomass and / or its extracts) used in accordance with the invention is superior; it is therefore preferred because it leads to optimal protection and without reaction at the injection site, or else to a reaction which is only mild and of short duration, whereas the adjuvants of the prior art have tendency to give rise to granulomas at the injection site. It will be understood that the foregoing description as well as the examples should be considered only as embodiments of the invention and it will be obvious, for those versed in the art, that modifications and variations can be made here without deviate 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 strain and / or subculture of H. pleuropneumoniae mentioned specifically. This is why the cultures obtained from this strain and / or subculture by natural and / or artificial selection and / or by mutation, as well as the cultures of any one of the various serotypes, are also within reach. of the present invention. CLAIMS 1. Vaccine to combat the infectious disease pleuropneumonia in pigs, a disease caused by the microorganism Haemophilus pleuropneumoniae, which comprises cells, cell parts, extracts and / or metabolic products of H. pleuropneumoniae as active ingredient and which contains adjuvants and a buffer, characterized by a content of a Bordetella pertussis vaccine, a biomass of B. pertussis and / or its extracts as an adjuvant. 2. Vaccine according to claim 1, characterized by a content of Vaccinum pertussis prepared, identified and tested in accordance with the European Pharmacopoeia, Vol. III (1975), 409, as an adjuvant. 3. Vaccine according to any one of the claim: 1 and 2, characterized by a Vaccinum pertussis content at 16 I.O.U. (international opacity units). 4. Vaccine according to any one of claims 1 to 3, characterized by a content of H. pleuropneumoniae cells, in parts of these cells, in its extracts and / or metabolic products, sufficient to produce a density of 20 I.O.U. 5. Vaccine according to any one of the claims - <EMI ID = 80.1> H. pleuropneumoniae cells per ml. 6. Vaccine according to any one of claims 1 to 5, characterized in that it has the composition: <EMI ID = 81.1> 7. Method of aerobic fermentation of the microorganism Haemophilus pleuropneumoniae in a liquid fermentation medium comprising assimilable sources of carbon and nitrogen as well as suitable salts and other nutritive ingredients conventionally used in the fermentation of microorganisms, in particular of the Haemophilus species, characterized in that one ferments in a medium comprising casamino acid, extract <EMI ID = 82.1> dinucleotide (NAD) as essential ingredients. 8. Method according to claim 7, characterized in that it ferments in a liquid medium having the composition: <EMI ID = 83.1> Water treated by ion exchange to make up to 1000 ml. 9. Method according to any one of claims 7 and 8, characterized in that fermented with stirring of the fermentation medium during the fermentation. 10. Method according to any one of the claims. <EMI ID = 84.1> substantially constant in the fermentation medium during fermentation. 11. Method according to any one of claims 7 to 10, characterized in that a pH of approximately 6.0 to approximately 7.9 is maintained in the fermentation medium. 12. Method according to any one of claims 10 and 11, characterized in that a pH of about 6.9 to about 7.5 is maintained in the fermentation medium. 13. Method according to any one of claims 10 to 12, characterized in that a pH of about 7.1 to about 7.4 in the fermentation medium. 14. Method according to any one of claims 10 to 13, characterized in that a pH around 7.3 in the fermentation medium. 15. Method according to any one of claims 7 to 14, characterized in that it is fermented in a medium having a substantially neutral initial pH. 16. The method of claim 15, characterized in that it ferments in a medium having an initial pH <EMI ID = 85.1> 17. Method according to any one of claims 7 to 16, characterized in that it is fermented in a medium having an oxygen concentration of about 1 to about 30 ppm during the fermentation. 18. The method of claim 17, character- <EMI ID = 86.1> from about 8 to about 20 ppm. 19. A method according to any one of claims 17 and 18, characterized in that it ferments at an oxygen concentration of about 8 to about 12 ppm. 20. Method according to any one of claims 17 to 19, characterized in that it ferments at an oxygen concentration of about 10 to about 12 ppm. 21. Method according to any one of claims 17 to 20, characterized in that the desired oxygen concentration is maintained by varying the stirring speed and / or the air flow rate. 22. The method of claim 21, characterized in that it ferments at a stirring speed of about 200 to about 600 revolutions / minute. 23. Method according to any one of claims 21 and 22, characterized in that it ferments at a stirring speed of about 500 revolutions / minute. 24. The method of claim 21, characterized in that it is fermented under aerobic conditions maintained using an air flow rate of up to about 0.5 liters of air per minute and per liter of fermentation medium . 25. The method of claim 24, characterized in that it ferments at a flow rate of air from- <EMI ID = 87.1> liter of fermentation medium. 26. Method according to any one of claims 24 and 25, characterized in that it ferments at an air flow rate of about 0.2 to about 0.3 liters of air per minute per liter of fermentatic medium 27. Method according to any one of claims 7 to 26, characterized in that it ferments at a <EMI ID = 88.1> 28. Method according to any one of claims 7 to 27, characterized in that a subculture of the H. pleuropneumoniae ATTC strain is fermented 27089 (NCTC 10976). 29. Process according to claim 28, characterized in that the 4226 subculture (State Veterin, ry Sérum Laboratory, Copenhagen, Denmark) of the strain is fermented. H. pleuropneumoniae. 30. Substrate for cultivating microorganisms, particularly suitable for cultivating the microorganism Haemophilus pleuropneumoniae, comprising assimilable sources of carbon and nitrogen as well as suitable salts and other nutritive ingredients conventionally used in the culture of microorganisms, in particular of the species Haemophilus, characterized by a content of casamino acid, yeast extract, glucose and beta-nicotinamide-adeninedinucleotide (NAD) as essential ingredients. 31. Substrate according to claim 30, particularly suitable for aerobic fermentation of H. pleuropneumoniae in a liquid fermentation medium, characterized in that it has the composition: <EMI ID = 89.1> 32. Substrate according to any one of claims 30 to 31, characterized in that it has been adjusted at a pH of around 7.2. 33. Substrate according to claim 30, particularly suitable for the culture of H. pleuropneumoniae on a solid substrate, such as for example on agar plates, characterized in that it has the composition <EMI ID = 90.1> 34. Substrate according to claim 33, characterized in that it has been adjusted to a pH of approximately gentlemen, <EMI ID = 91.1> Referring to the above request, our <EMI ID = 92.1> some material errors in the memo: description: - page 5, line 10, it should read: "Nielsen" - page 19, sample no 7, the oxygen concentration! (ppm) is 7.2 <EMI ID = 93.1> attached to the file of an invention patent cannot be such as to bring either to the description or to: substantive changes and declares that the content of the note does not make such changes and is only intended to report one or more material errors It acknowledges that the content of this cannot have the effect of making it totally or <EMI ID = 94.1> <EMI ID = 95.1> It authorizes the Administration to attach a note to the patent file and to issue a photo, <EMI ID = 96.1> distinguished.