CA1189790A - Process and a substrate for the aerobic fermentation of haemophilus pleuropneumoniae - Google Patents

Abstract

Abstract of the invention In an improved method for combating pleuropneumonia in pigs by administering a vaccine comprising cells of the known and generally available microorganism Haemophilus pleuropneumoniae, parts of such cells, extracts and/or me tabolism products thereof as the active ingredient, adjuvants and a buffer, the improvement consisting in the use of a sor-detella pertussis vaccine, biomass and/or extracts thereof as adjuvant, as well as the new H. pleuropneumoniae vaccine comprising a B. pertussis based adjuvant. The method and the vaccine result in an improved protection of the pigs against pleuropneumonia attacks without side-effects. Be-sides, a new and improved substrate called CAY-substrate for the cultivation of microorganisms, in particular the bacterium H. pleuropneumoniae, which compared to known sub-strates results in a substantially higher yield of biomass suitable for the production of a H. pleuropneumoniae vac-cine, said substrate comprising casamino acid, yeast ex-tract, glucose and NAD as essential ingredients and being suitable for cultivation of H. pleuropneumoniae on solid as well as in liquid media by slight modifications in the composition of the substrate An improved process for the aerobic fermentation of H. pleuropneumoniae in a liquid medium comprising the new CAY-substrate, the fermentation being advantageously performed at a temperature of about 37°C., a pH of about 7.1 to 7.4, and at an oxygen concen-tration in the fermentation medium of about 8 to 12 ppm, thereby maintaining the desired oxygen concentration by variation of the stirring speed and/or of the air flow rate.

Description

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Background of the Invention In the early 1960's Haemophilus-like bacteria were isolated in Great Britain, California and Argentina from herds of pigs which were attacked by pleuropneumonia.
It was the bacterium Haemophilus parahaemolyticus - or Haemophilus pleuropneumoniae as the Argentine strain was called - which was ihe cause of the disease pleuropneu-monia. Since then attacks of the disease have been reported in many countries, including Denmark, cfr. R. Nielsen, Nord. Vet. Med. 22 (197~), 240-245.
The disease has a very acute course and is often accompanied by a high mortality and since it appears rather frequently in many herds of pigs, some efforts have been made in an attempt to develop a vaccine which would protect t~e pigs against the disease. A common feature of these vac-cines is their use of killed cells of H. pleuropneumoniae and in most cases also an addition of an adjuvant.
Vaccination tests have also been made under field conditions, cfr. R. Nielsen, Nord. Vet. ~led. 28 tl976), 337-348. In these tests 6 or 24 hours old agar plate cultures of H. pleuropneumoniae were used as an antigen. ~rne cells were killed by formaldehyde and the vaccine contained 101 cells per ml. in phosphate buffered saline (physiological) (in the following for short PBS) containing 0.2~ formaldehyde and with an adjuvant added. The vaccine was administered as subcutaneous injections of 2 times 4 mls. or 2 times 2 mls.
with an interval of 14 days. The first vaccination was made when the pigs were 9 weeks old. 2 weeks after the second vac-cination the pigs were infected with 101~ H. pleuropneu~oniaebacteria. As adjuvants 15% Alhydrogel (aluminium hydroxide gel) and Freund's Incom~lete Adjuvant/ cfr. J. Freund, Am.
~ev. Microbiol. 1 ~1947), 291, resnectivelv, wére llsed.
; The results of these vaccination tests are summa-rized in the following Table I.

7~

Table I

Aqe of anti en Ad'uvant Protection (%) g l ,, 6 hours 15% Alhydrogel 67 Freund's Incom~l. __ 90 24 hours 15~ Alhydrogel 25 Freund's Incompl._ _ 67 .. . . _ . _ _ _ _ It should be added that the use of Freund's Incom-plete Adjuvant gave rise to a formation of granulomes at the injection site.
Atte~pts have also been made in order to obtain a protection by using 20 hours cultures killed by formaline as the antigen and Alhydrogel and Freund's C~lete~d~uv~nt, respectivelv, as the adjuvant. The dosis injected was 2 ~les 5 mls. containing 109 bac-teria per ml. The first injection was made intramuscularly on 50 days old pigs and the second injection 1 week later. 1 week thereafter, the Pigs were in~ected. The result was a significant evidence ~hat the vac-cine had a protecting effect, but no significant evidencethat one adjuvant had advantages above the other.
~ E. Scholl et al., cfr. Proc. Int. Pig Vet. Soc. Con ; gress, Ames, Iowa, U.S.A. (1976), have used heat killed cul-tures of H. pleuro~neumoniae as antigens and have vaccinated by two methods: 1) by increasing doses, viz. 0.5, 1 and 3n~s., and-2) by 2 times 5 mls. All injections were made with an interval of 2 weeks. Serological test of blood samples showed that repeated'injections of small doses resulted in anti-bodies in the blood, but no sensitized lymhocvtes, whereas injections oX high doses resulted in sensitized lvmphocytes, but no antibodies in the blood.
The same workers, cfr. Proc. Int. Pig Vet. Soc.
Congress, ~agreb, Yu~oslavia (1978), have used cultures of H. pleuropneumoniae killed by formaline in concentrations of 109 bacteria per ml. The vaccination doses and the methods o~
- vaccination were the same as in the earlier reported work, ~3~

but in this later work a group of placebo pigs was included and the animals were put into a chronically infected herd of pigs. Clinical symptoms were observed and following the slaughtering the lungs and the thoracic cavity ~rere ex~ned.
The results showed a good effect of the vaccination, but no differences between the two methods of vaccination.
M.F. de Jong, cfr. Proc. Int. Pig Vet. Soc. Con-gress, Zasreb, Yugoslavia tl978), has made vaccination tests with 24 hours H. pleuropneumoniae cultures to which an in-complete adjuvant on the basis or mineral oil had been ad~ed.For each dosis 20 mgs. (wet weight) antigen were used and two injections were made with an interval of 3 weeks, the iirst vaccination when the pigs were 10 ~eeks old~ 3 weeks after the second vaccination, the pigs were challenged with 15 or 24 hours cultures. With 24 hours challenge cultures, the pigs became ill, but recovered within 1 ~eek. On the other hand, ~he pigs challenged with a 16 hours challenge culture died within 36 hours. Challenge involves, as will be known by those skilled in the ar~, an ex~erimental infection of the test animals with live cells of the infectin~ micro-organism.
~ M~F. de Jong, loc. cit., then used 6 hours cultures ; ~or ~oth vaccination and challenge. The pigs were challe~yed with different amounts of cells and the mortality rate was dependent upon the number of cel]s used. Besides, ~igs having ~een vaccinated with 6 hours cultures were challenged with 16 hours cultures and 50~ of the pigs survived. lwee~ later, the survivin~ pigs were infected with a 6 hours culture and now they did not show even sym~toms of illness The works on test vaccinations mentioned abov~ clearly show that it is necessary to add an adjuvant to a vaccine com~rising H. pleuropneumoniae antigens in order to obtain a sa*isf2ctory effect of the vaccination. The substances most commonly used as adjuvants are Alhydrogel and Freundls Adju ~ 35 vant (Comlete or In~omDlete~ Of these substances, Freund's ; Adjuvant has until now been the most efective, but as also ''3"7~

reported, cfr. R. Nielsen, loc~ cit. (1976), it results inthe formation of granulomes at the injection site, and ~here-fore it is considered unsuitable. Thus, the problem of pro-viding an adjuvant which is effective, but does not inter-fere with the animals has not been solved by the previouslyreported test ~accinations.
Another unsolved problem is the preparation of an antigen for the production of the vaccine. This ~roblem re-sides in the fact that H. pleuropneumoniae has a rather poor growth on the ~reviously used and classical substrates which sets strongly restricted limits ~or the amount of vaccine which it has been possible to produce.
Thus ~. Nicolet, cfr. Zbl. Bakt. I Abt. Orig~ 216 (1971~, 487-495, in his serological studies has used a SU'D-tra~e comprising PPLO-agar (cfr. DIFCO-Manual IX, 1953t Catalogue No. 412 - PPLO = pleuropneumoniae-like or~anisms) ; to which had been added 0.1% glucose, 2.5~ yea~t extract, 5~ horse ser~m and 5 mgs./liter NAD (~-nicotinamide-adenine--dinucleotide). The same substrate was used by R. Nielsen, ~0 loc cit. (1976).
M.F. de Jong, loc. cit., has used Brain Heart Infu-sion CQn~aining NAD as the substrate, and M. Kilian et al., cfr. Int. J~ Svst. Bacteriol. 28 (1978), 20-26, have made taxonomical studies of H. pleuropneumoniae, thereby culti-~ating the strains on a substrate comprising:
1% of Neutralized Bacteriological Pe~one ("Oxoid") 1% of Yeast Extract 1~ of Glucose, - 10 mgs./liter of NAD, and a salt solution as disclosed by L.V. Holdeman and W.E.C. Moore in Anaerobe Laboratory Manual~ 3rd ed., -Virginia Polytechnic Institute, Anaerobe laboratory, Blacksburg (1975).
A few other workers have cultivated H. pleuro~neu-moniae in liquid cultures, but with rather ~oor results.
' , ' ' .

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An objec-t of the invention is to ~rovide a new and improved vaccine for effectively controlling pleuropneumonia in pigs, thereby also providing a new and improved adjuvant for such a vaccine without the side e~fects associated with the prior art adjuvants.
Another object of the invention is to nrovide a new and improved fermentation process for the preparation of the antigen necessary for the production of the vaccine of this invention so as to make it possible to produce an effective va~cine in amounts sufficient for an extensive vaccination when desirable.
A further object of the invention is to provide a new and improved substrate for the cultivation of H. pleuro-2neumoniae so as to obtain a growth of the microor~anism which is substantially better than when cultivating on prior art substrates.
A still further object of the invention is to provide a method for combating pleuropneumonia in pigs by admi~
istering the vaccine of this invention.
These and other objects are obtained by providing a vaccine which comprises cells of Haemophilus pleuxopneu-moniae, parts of such cells, extracts and~ox metabolism pro-ducts thereof, obtained by cultiva.ing a strain of H. ple~^o-pneumoniae in a liquid culture based upon the substrate dis-closed by S.MD Cohen and M.W. Wheeler in Am, J. Publ. Hlth.
36 (1946), 371-376, said vaccine also com~rising as an ad-juvar.t aluminium hydroxide gel and/or Freund's Incomplete or Complete Adjuvant and/or Bordete~lla Dertussis vaccine,bi~.~ss and/or extracts thereof, as ~7ell as a sterile puffer solu-tion as a diluent.

~ ~a ~ 9~

Detailed_Description of the Invention The bacterium used for the production of the vaccine of this invention is a strain of Haemophilus pleuro~neumoniae which is a known microorganism deposited in various culture collections, cfr. below~ and available therefrom.
Investigations have shown ~hat there exist at least 6 different sexoty~es of H. pleuropneumoniae, cfr. M. Kilian et al., loc. cit. (1978). This literature reference also ex-plains why the name H. pleuropneumoniae is considered thecorrect name of the organism causing pleuro~neumoni~ in pigs and gives a general survey of the characteristics of the microorganism.
- Of the various serotypes of H. pleuropneumoniae de-scribed in the above literature reference, some have been deposited, thus serotype-1 in the American Type Cultur~ Col-lection, Rockville, Md., U.S.A., under No. ATCC 2708~, sero-type 2 under No. ATCC 27089, and serotype 3 under No. ATCC
27090. Besides, serotype 2 has been de~osited in the ~ational Collection of Type Cultures, Colindale/ London, U.~., under No. NCTC 10976, and these three serotypes have all been de-posited in the Czechoslovak Collection of Microorganisms, ~rno, Czechoslovakia, under the Nos. CC~ 5869, CCM 5870, and CCM 5871, respectively.
~loreover, M. Kilian et al., loc. cit. (1978), de~
s~Frihe serotypes desigI-ated as serotyPes 4, 5, and NT. ~low-ever, no deposition numbers are given.
Applicant's own investigations have confirmed that H. pleuropneumoniae bacteria are small Gram-ne~ative immo-bile rods which may take a number of various shapes, dependeNt - UpOII the culture substrate used and the environmental condi-tions.
Thus, on agar plates they are most often small coc-coidal or short rods whereas in liquid cultures they may 35 ~ary from small cocco dal rods to longer slender or thic~ -rods, as an exception (which is rather seldom) even to pleo-- morphical ilament shapes.
.

Cultured on agar plates on the substrate of this .invention (composition, cfr. Table ~II below), H. Pleuro-pneumoniae bacteria are shaped as small, coccoidal rods, and the individual colonies have a diameter of up to 3 mms.after 48 hours at 37C. Fermented in the nutrient broth of this invention (composition, cfr. Table IV below), H. ~leuro-- pneumoniae bacteria are short rods which are often inshorter or longer chains.
In biochemical respect, H. pleuropneumoniae bacte-ria behave as described by ~-. Kilian, J. Gen. Microbiol. 93 (1976), 9-62, having the following characteristic properties which together make them different from other Haemo?hilus s~ecies: V-factor-dependent, able to svnthesize por~hyrine - from delta-aminolevulinic acid, produce urease and able to ferment mannitol, xylose and desoxyribose.
~ lore specifically, a strain of H. ~leuropneumoniae has been used which was isolated from a ~ig having pleuro-~neumonia. This strain was of serotYpe 2, cfr. R. Nielsen, loc~ cit (1976) ~ and was submitted to Statens Veterinaere Serumlaboratorium (State Veteri~ary Serum Laboratory),Co~en-hagen, Denmark, in which it was given the desi~nation Suh culture 4226n The substrate used in the ~ractice of this inv~ lon is based upon the substrate disclosed by S.M. Cohen and M.W. Wheeler, loc. cit., having the composition given in Table II below.

37~1~

Table _ Casamino acidX)~ technical 10.0 gs.
NaCl 2.5 gs.
KH2PO4 0 5 g M~C12 6H2 0.~ g.
Soluble starch 1.5 gs.
CaC12 (1% solution) 1.0 ml.
FeSO~ 7H2O (0.5% solution) 2.0 mls.
10 CuSO4 5H2O (0.05~ solution) 1.0 ml.
Cystein HCl (1% solution) 2.5 mls.
Yeast dialysate 50.0 mls.
Distilled water to 1 liter pH adjusted to 7.2 7.3 Difco Manual 1953, No. 231, "Bacto"

For the purposes of this invention, this substrate ; is modified so as to improve the growth of H. pleuropneu-moniae and provide a substrate which is suitable for the pre-p~ration of a H. pleuropneumoniae vaccine in amounts su~fi ; cient for extensive vaccination, if desired. Twodifferent mct - difications are used for cultivation on agar plates and fer-mentation in liquid medium, res~ectively.
For agar plates J a substrate comprising casamino acidt ye~st extract, glucose and ~-nicotinamide-adenine-di-nucleotide (in the following called NAD) as essential ingre-dients is used. Besides, this substrate should contain suit-able salts and other nutrient ingredients conventionally used when cultivating microorganisms, in particular Haemo~hilus species. An especially preferred substrate has the composi-tion given in Table III below.
.

' Table III

Substrate for agar plates 5 Casamino acid, technical (Difco Manual) 6.0 gs.
Yeast extract, technical (Difco Manual) 25.0 gs.
Glucose tPharmacopoea Nordica) 2.0 gs.
NaCl 2.5 gs.
H2P4 0.5 g.
MgC12 6H2 0.l g.
CaC12 (1% w/v solution) 1.0 ml.
FeSO4^7H2O (0~5% w/v solution, freshly prepared) 0.5 ml.
Cystein HCl (1% w/v solution) 0.8 ml.
Agar-agar (Di~co Manual) 16.0 gs.
15 ~AD (2.5% w/v solution) 1.0 ml.
~Sigma Chemical Co., St. Louis, Mo., U.S.A.) Water to 1000 mls.

; 20 This substrate is prepared in the followingr~lcr:
Casamino acid, yeast extract, glucose, NaCl, ~l2P~
and MgC1~ 6H2O are dissolved in 950 mls. ion-exchanged H2O.
p~ is adjusted to 7.2 by the addition of 1 N NaOH. CaC12, FeSO4 7H2O, cystein HC1, and agar-agar are added and the volume is adjusted to 1000 mls. by the addition OL ion-ex-changed H~O. The solution is heated to boiling (ln or~er to - dissolve the agar), autoclaved at 121C. and a pressure of ~2 atmos~heres for 15 minutes. Ater cooling to 50C~, the ~AD is added and the substrate is poured into Petri dishes.
The substrate OL this invention for use in the fermentation in a liquid medium also comprises casamino acid yeast extract, glucose and NAD as essential ingredients.
This substrate too should contain suitable salts and o~her nutrient ingredients commonly utilized in the fermenta~ion of microorganisms,in particular Haemophilus species. An espe-cially preferred substrate has the composition given in Table IV below.

Table IV

Substrate for fermentation in liquid medium 5 Casamino acid, technical (Difco Manual)18.0 gs.
Yeast extract, technical (Difco Manual)14.0 gs Glucose ~Phar~aco~oea Nordica) 11.0 gs.
NaCl 2.5 gs.
KH2 4 g 10 MgC1~ 6~20 - 0.1 g CaC12 (1~ w!v solution) 1.0 ml.
FeS04 7H20 (0.5% w/v solution, freshly prepared) 2.0 mls.
CuS04 5H20 (0.05% w/v solution) 1.0 ml.
Cystein-HCl ~1% w/v solution) 2.5 mls.
15 l~AD (2.5% w/v solution)(Sigma Chemical Co.) 1.0 ml. ;
Water to 1000 mls~

This substrate, called CAY-substrate for short, is preDared in the followin~ manner:
Casamino acid, yeas-t extract, glucose, NaCl,K~zPO~, and ~IgC12 6H~0 are dissolved in 950 mls. ion-exchanged H20.
p~ is adjusted to 7.2 by the addition of 1 N NaOH. CaC12, FeS0~ 7H20, CuS04 5H20 and cystein HCl are added, the volume is adjusted to 1000 mls. by the addition of ion~exchan~ed 25 H20, and the solution is autocla~ed at 121C. and a ~ressure of 2 atmospheres for 15 minutes. After cooling, NA~ is added and the substrate is ready for use.
Both of the substrates disclosed in Tables III and IV com~rise assimilable sources of carbon and nitrogen which can be u~ ed by the microorganism ~. ~leuropneumoniae. On both substrates, an abundant and dense gro~Jth of the organism is obtained.

7~

The fermenta-tion procedure is as follows.
The strains of bacteria are stored in a lyophil-ized state in ampoules prepared by one of the methods de-scribed below. The storage is effected in a refrigerator at 5C.
Method ~. The bacteria were cultivated at 37C.
for 18 hours on agar plates with a su~strate having a com position as set out in Table III. The cells were harvested by the addition of 1 ml. PBS (composition given in Table V
below) and subsequent abrasion of the cells. The cells were transferred to ampoules which were immediately cooled to -25C. and then transferred to a lvo~hilizer and lycphilized.

Table V
Composition of PBS

NaCl 8.0 gs.
KCl 0.2 g~
Na2HPO4 1.15 gs.
KH2PO4 0.2 g.
Distilled water to 1 liter.
.

The salts are dissolved in distilled water and autoclaved at 121C. and a pressure o~ 2 atmospheres for 15 minutes. pH is 7.3.
Method B. This method was similar to Method A, hcw-ever 1 ml. skim-milk was used for each ~late instead of PBS.
The skim-milk had been heat-treated by heating to 100C. for 15 minutes on 3 subsequent days.
Method C. The bacteria were cultivated for 6 hours on a water-bath in the CAY substrate, cfr. Table IV. The fully developed suspension of bacteria was centrifuged and the bacterial cells were resuspended in sterile skim-mil~
and transferred to ampoules which were cooled and lyophil-ized as described for Method A.
, ~, 7~) The bacteria are restored ~rom the lyophilized ampoules by the addition of substrate followed by inocula-tion on agar plates and in tubes containing li~uid sub-strate.
The living cultures are maintained by a daily inoculation on fresh agar plates containing the suDstrate of Table III. The plates are incukated in an incubator at 37C.
A Preculture is prepared by propagation of tle bac-teria. This is done by inoculation from an agar plate into a flask containing 50 mls. of the liquid CAY-substrate.
The flask is incubated in an incubator at 37C. for 8 nours.
10 mls. of medium is then ~ithdrawn for inoculation of the second preculture.
The second preculture is 500 mls. of the li~uid CAY-substrate in a 1 liter conical flask which is incuhated on a water-bath with continuous sha~ing. The temperature is 32C. and the incubation period is 18 hours.
The second preculture is used for the main fermen-tation in ~t7hich the liquid CAY-substrate of Table IV is used as the fermentation medium. The medium is prepared as desc-ibed above for the CAY~substrate, i.e. by dissolving casamino acid, yeast extract, glucose, NaCl, KH2PO4 and 1~3gC12 6H2O in ion-exchanged water, adjus-ing the pH to about ? 2, adding the re~laining ingredients except for the N~, autoclaving the medium at a temperature of at least a~out 121C. and at a pressure of at least about 2 atmos-phexes for at least lS mi~utes, cooling to a temperature o not more ~han 37C., controlling and readjusting pH to about 7.2, and adding the NAD.
The ~ermentor is then inoculated with the second preculture of H. pleuropneumoniae obtained as described a~o~.
~uring the ~ermentation, a temperature of preferably about 37QC. is maintained and an air flo~7 throu~h the medium is 3~ used in order to maintain a suitable oxygen concentration in the medium~ This may be done partly by controlling the air flo~ rate and partly by controlling the speed of the stirring device of the fermentor, thereby taking into con sideration that these parameters are mutually dependent, in-asmuch as an increase in any of them results in an increased oxygen concentration whicn in turn may necessitate a de-~crease in the other parame,er.
For the fermentation of H. pleuropneumoniae, the oxygen concentration may vary from about 1 to about 3a ppm, the most suitable concentration being from about 8 to about 20 ppm, in particular from about 8 to about 12 ppm, more specifically ~rom about 10 to about 12 ppm. The air flow rate and stirring s~eed necessarv in order to maintain such an oxygen concentration will of course de~end upon the dLmen-ions, equipment and arrangement of the fermentor, but it has been found that an aix flow rate of u~ to about O.~ er of air per minute per liter of rermentation medium, in par-ticular from about 0.15 to a~out 0 4 liter, more specifically from about 0.2 to about 0~3 liter, of air per minute per liter fermentation medium is most suitable in a fermentor having a capacity of about 20 liters. For this fexmentox, a suitable stirring speed is from about 200 to about 600 r.p.m., in particular about 500 r~p.m.
It should be emphasized that these parameters are not critical in the practice of ~his invention~ and for any fermentor those skilled in the art will be able to adjust the parameters so as to main.ain the desired oxygen concen-tration.
During the fermentation it is ~referable to main-ta{n a substantially constant pH. A suitable pH is in the range of from a~out 6.0 to about 7.9, in particular from about 6.9 to about 7.5, more specifically from about 7.1 to about 7.4, preferably at about 7.3. This may be accom~lished by the addition of a hase, suitably an aqueous sodium hydroxide solution, when necessary.
It is not preferred to add an anti-foam agent, ~ut it may be necessary to do so. Any conventional anti-foam -37~

a~ent for use in fermentation processes is suitable for this purposeJ in particular a silicone anti-foam agent.
The fermentor is preferably provided with means which allo~ an automatical addition of pH-controlling base and/or anti-foam agent during the fermentation. ~o~ever, the addition may also be accomplished manually, ~hen neces sary.
During the fermentation, samples are withdra~7ninter-mittently and used for a determination OL the cell density.
The density was measured by a "Corning Colorimeter 252" at a wave length of 600 nm and expressed as number of colony ~or~ing units (CFU) by means of a reference standard curve.
This density measurement serves the purPose of drawing a growth curve which allows the interruption of the fermenta-1~ tion at the time most suitable for the subse~uent productionof a ~accine, i.e when the phase in which ,he bacteria show an ex~onential growth has ex~ired.
I~en the fermelltation has been com~letecl, a con-centrated formaldehyde solution and an Alhydrogel solution are added to the fermentation medium and after stirring for a substantial time, ~ over night, the suspension is sub-jected to a centrifugation resulting in a formaldehyde-con-taining supernatant and a precipitate comprising H. pleuro-pneumoniae cells and Alhydrogel. The precipitate iscollected in a sterile vessel. A sample is withdrawn for controlling that the bacteria have been killed.
The following Exam~les further illustrate the ad-vantages ob ained by cul~ivating H. pleuro~neumoniae on the CAY-substrate of the present invention.
ExamDle 1 This Example sho~7s the fermentation of Haemophilus pleuropneumoniae, strain ATCC 27089r Subculture 4226, on the CAY-substrate of this invention.

37~

The fermentor was a "Biomat E 20" (Moell~r &
Jochumsen, Vejle, Denmark) having a capacity of containing 20 liters substrate in each fermentation and being equi~ped with means ~or stirring, said means allowing a manual varia-tion of the stirring speed, an instrument which continuouslyshows -the stirring speed (r.p.m.), means forsupplying sterile air, means for a continuous measurement and recording of the oxygen concentration of the fermentation medium, means for a continuous measure~ent and automatic ad~ustment of the temperature to a pre-determined value, means for a con~inuous measurement and recording of the pH o~ the fermen~ation n~dium and for manual or automatic addition of pH-controlling bases or acids, means for controlling the foaming in the fermentor and for the automatic addition of anti-foam agent, when ne-1~ cessary, and means for the withdrawal of samples during thefermentation.
Tne fermentor was charged with 17 liters ion-ex-changed water and stirring was started. Then 306 ~s. cas-amino acid, 238 gs. yeas, extract, 187 gs. glucose, 42.5 g5.
NaCl, 8.5 gs. KH2PO4, and 1.7 gs. MgC12 6H2O were added.
- p~ was adjusted to 7.2 by the addition of a 1 N NaOH solu-tion and 17.0 mls of a 1 ~ w/v solution of CaC12, 34.0 mls.
of a 0.5~ w/v solution of FeSO4 5~2O (freshly pre ared~, 17.0 mls. of a 0.05% w/v solution of CuSO4 5H2O, &nd 42.5 mls.
of a 1~ w/v solution of cystein HCl were added. The fenmentor was closed, autoclaved for 15 minutes at 121C. and a pressure of 2 atmospheres and then cooled to 36C. Then 17~0 mls. of

2.5~ w/v solution of ~IAD were added. This ~rocedure resulted in a fermentation medium having the com~osition given in Table IV and with a pH of 7.45.
The fermentor was inoculated with a 600 mls. second preculture of H. pleuropneumoniae prenared from a lyophili-ed bacteria stock and cultivated as described above. The fe ~ n-tation period was 7 hours and 50 minutes and samPles were intermittently withdrawn and used for determining the absolu~e cell density of the fermentation medium. The density was ~.

~ t97 measured on a "Corning Colorimeter 252" at a wave length of 600 nm and then converted into cells per ml. by using a standard reference and a conversion table which made it ~os-sible to draw a growth curve and to determine when the ex-ponential gro~Jth phase of the bacteria had come to an end.
This fermentation was carried out ~rimarily as an experimental fermentation and therefore it was allovied to ~roceed in a some~Jhat "zbnormal" manner, which involved that the pH was not adjusted during the fermentation and that no anti-foam agent was added.
The results obtained from analyzing the samples are given in Table IV below.
It will be seen that the oxygen concentration ~hich preferably snould be 8 to 10 ppm decreased drastically d~ring the ~exmentation, in particular in the exponential growth phase. This was counteracted by increasing partly the air flow rate and partly the stirring speed. It will also be seen that pI3 decreased to a final ~H of 5.30.

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U~ I , The results in Table VI have been used for draw-ing the growth curve shown in Fig. 1 of the drawings. In the figure, the abscissa shows the time after inoculation, and the ordinate shows the number of cells per ml. fermen-tation medium. It should be noted that the ordinate is de-picted on a logarithmic scale. The figure shows that the ex ponential growth phase ends in a~out 5 hours after the ino-culation which means that the fermentation should have been stopped at this point if the medium had been intended for use in theiproduction of a vaccine. However, as this ~er mentation was an experimental fermentation, it was continued into the stationary growth phase.
Fig. 1 of the drawings also makes it possible to calculate the time (Tg) necessary for doubling the contents of bacteria in the fermentation medium during the exponential growth phase. Tg is given by the formula loge Tg = ~
lu wherein ~ is represented by the ~ormula u = ge C2 loge Cl (II) T2 ~

wherein C2 is the number of cells per unit volume at the time T2 after inoculation, and Cl is the number of cells per unit volume at the time T~ after inoculation.
In Fig. 1 the exponenti~l growth phase is repre-sented by the straight line part of the curve. Thus, it will be seen that this phase extends from not later than 1 hour to about 5 hours after inoculation. Using this part of the curve (the results marked with a circle in Fig~ 1, i.e.
samples Nos. 6 to 18) for a calculation according to formula (II~ shows that

3~0 u = 3.27 + 0.92 = 1 05 wherein T2 and Tl have been expressed as hours and the com-mon factor 10 has been ignored in the calculation of loge C2 and loge Cl. According to formula (I) this means that Tg = 0.693 = 0.660 hours - 1.05 which equals 39.6 minutes.

Example 2 This ~xample shows a "normal" fermentation which involves that pH is continuously and automatically ad]usted to about 7.2, that anti-foam agent is automatically added when necessary, that a moxe vigorous stirring is~ntained, and that the oxygen concentration is continuously held at a relatively high level.
- The fermentor was identical to the fermentor used in Example 1. The volume of the fermentation medium was about 20 liters of which the inoculation medium provided 0.5 liter, the balance being lg liters ion-exchanged water containing casamino acid, yeast extract, glucose and salts in amounts providing a CAY-substrate of the relative composition given in Table IV. The charging of the fermentor 7 the autoclaving, cooling and subsequent addition of NAD was carried out as de-scribed in Example 1. The initial pH of the medium was 7.1 and the temperature at the inoculation was 31C.
When the inoculation medium had been added, the fer-mentation was started and continued for 7 hours 30 minutes.
; The inoculation medium was 500 mls. of a second preculture o~ ~. pleuropneumoniae,strain ATCC 27089, Subculture 4225 r cultivated in the manner described above.
;, ~. -7~

~ uring the fermentation the pH was maintained at an almost constant value of about 7.2 by the auto~
matic addition of a 1 N Na~H solution when necessary.
Stirring was performed at 500 r.p.m. and the oxygen concentration was maintained at from about 8 to about 12 ppm. This was achieved by increasing the air flow rate when necessary~ An anti-foam agent known under the name "Silicone RS Antifoam C. DAK" was automati-cally added when necessary.
Samples were withdrawn during the fermentation and analyzed as described in Example 1 in order to follow the growth rate. The fermentation period was 7 hours 30 minutes.
The results of the fermentation have been sum-mari~ed in Table VII.

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The data given in Table VII have been used for dr~-ing the growth curve shown in Fig. 2 of the drawings. The abscissa and the ordinate have the meanings givenfor Fig. 1, cfr. Example 1. From Fig. 2, it will be seen that the fer-mentation is stopped while the microorganism i5 s*ill in theexponential growth phase although the growth rate is some-what less pronounced ~han in the initial phase of ~e re~len-tation, a slight decrease in the slope of the curve occ~-ring at about 3 hours 30 minutes after the inoculation.
The measurements shown with circles in Fig. 2 have been used for calculations according to the formulae (I) and (II) of Example 1, partly for the initial phase;(phase 1, samples Nos. 5 to 13), and partly for the last period (ph~e samples Nos. 15 to 27) of the fermentation. The units used in the calculations in Example 1 were used in these calcula-tions, too, which gave the following results.

., ~ul = 1 19 ~ 1 05 = 0 914 h~
3.25 - 0.8 Tgl _ 0.693 = 0.758 hour 0.914 ; which equals 45.5 minutes.

3.62 - 1-70 = 0 s4g h 7.5 - 4 Tg2 = 0-69 = 1.26 hours 0.549 which equals 75.7 minutes.
~ comparison of the results obtained according to Examples 1 and 2 will show ~hat although the "abnormal" ~er-mentation process of Example 1 results in the lowest initial Tg, which means a fast~r growth of the bacteria in the fer-;

7~

1, mentor and consequently a shorter fermentation period before the broth i5 ready for utilization for the production of a H. pleuropneumoniae vaccine, it also results in a faster ~r~-ition into the stationary growth phase, viz. after a fermen-tation periodof about 5 hours, whereas the "normal" fen~.~tionresults in a prolonged period of exponential growth phase of the bacteria, viz. more than 7 1/2 hours. More important, the "normal" fermentation also results in a higher yield of cells per unit volume at the time where the broth is ready for vaccine production, viz. more than 37 x 108 cells per ml. compared to about 27 x 108 cells per ml. in Example 1 (sample No. 18 where the exponential growth.phase ends).~ere-fore, from a commercial point of view (vaccine production) the "normal" fermentation process of Example 2 is preferred because the greater time consumption is more than counter .balanced by the higher yield of cells utilizable in the sub- ¦
sequent vaccine production.

Example 3 This ~xample is a Comparison Examp1e ~lic~ldemonstrates that fermentation of H. pleuropneumoniae, Subculture ~226, on a kno~n substrate results in a much slower growth rate and a much. lower yield than fermentation in the substrate of this invention~
The fermentor was identical to the fermentor used in Examples 1 and 2.
The substrate was a commercial "CASO-bouillon Merck"
which is described as No. 5478 in "Handbook of Microbiology"
published by E. Merck, Darmstadt, FederalP~public of Germany, to which 6 mgs.NAD per liter bouillon had been added.~e fer-mentor was charged with 17 liters of CASO-bouillon, auto claved and cooled to 37C~ as described in Example l,and then NAD was added. The initial pH was 7.3 As inoculation medium, 600 mls. of a second pre-culture of H. pleuropneumoniae ATCC 270gg,~SuDculture 422~, w~
.

added. This preculture had been cultivated in the manner described above. During the fermentation, a temperature of 37C. was maintained. pH was controlled manually and allowed to decrease to a final pH o:E 6.85. The initial oxygen concen- ' 5 tration was 10 ppm~but it dropped very fast to 0, partly ', because the air flow rate is low and partly because ~le stir-ring was stopped immediately after the inoculation and not resumed until 1 hour after an oxygen concentration of 0 had been reached.
Thus, it is characteristic for this fermentation that it must take place under almost anaerobic conditions in order to obtain an acceptable yield which ne~ertheless is low, cfr. Table VIII below.
The fermentation period was 6 hours 35 minutes and during this period samples were withdrawn and analyzed in the manner and for the parameter described in Example 1.
The results of the fermentation are summarized in Table VIII.

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The results oE Table VIII have been used for draw-ing the growth curve shown in Fig. 3 of the drawings. The abscissa and the ordinate represent the same units as stated in Example 1 for Fig. 1. ~y a comparison of the curves in Fig. 1 and 2, respectively, and Fig. 3, it is clearly apparent that the growth rate of H. pleuropneumoniae is substantially faster in the CAY-substrate of this inv~ntion (Figs l and 2) than in the known CASO-bouillon (Fig. 3) and this is con-firmed by calculations according to the formulae (I) and (II~ in Example 1 and given below.
From Fig. 3 it will be seen that the ex~onential growth phase of the bacteria ends at about 5 hours after inoculation and that this Phase, as was the case in the fer- ¦
mentation of Exam~le 2, has two different Dhases of which phase 1 ends about 3 1/2 hours after inoculation, i.e. about the time where the resumed stirring results in an oxygen concentration above 0 although this period is short.
The calculations made cover sam~les Nos. 3 to 14 (phase 1) and samples Nos. 14 to 21 (phase 2) and were made as described in Example 1. Again, the basis for the calcu-lation is the Points on the curve of Fig. 3 shown with circles. The results are the following ~1 = 1 ,0 = 0.533 h 1, 2.34 - 1-70 , 0 350 h 5.33 - 3.5 Tg = 0-693 ~ 1.30 h, 0.533 .1 Tg2 = 0-693 = 1.98 h, -¦
0.350 or Tgl = 78.0 minutes and Tg2 = 11~ minutes.
.j 1.

These results show that the exponential growth rate of H. pleuropneumoniae bacteria when fermented on the CAY--substrate of this invention is surprisingly faster than the fermentation on CASO-bouillon, viz. approximately twice as ast in phase l (Tg -- 39.7 and 45~5 minutes versus 78 min-utes) and that the growth rate in phase l on CASO-bouillon is even slo~Jer Lhan the growth rate in phase 2 of Example 2 on CAY-substrate ~Tg = 78 minutes versus 75.7 minutes).
Besides, the yields on CAY-substrate are surpris-ingly higher than the yield on CASO-bouillon ~6.4 x 108 cells per ml. in Example l at the end of the exPonential growth phase and 37.2 x 108 cells per ml. in Example 2 ver-sus 10.2 x 108 cells per ml. in Example 3). Thus, the yields on the substrate of this invention are about 2.6 and 3.6, respec~ively, times the yield on the known substrate.

E~-.am~le 4 ;

After a fermentation period of 7 1~2 hours, the ~ermentation described in Example 2 was stopped by the add~
ition of 60 mls. concentrated formaldehyde solution and 600 mls. of Alhydrogel. The Alhydrogel added was a 2~ w/v solution of aluminium hydroxide. After vigorous stirring o~ernight (for about 18 hours), the fermentation broth was transferred to a stora~e vessel~ from which it was pumped to a disc centriuge and sepaxated into an aqueous rormald~yde~
-containing supernatant which was discarded and a precipitate comprising H. pleuropneumoniae cells a~d Alhydro~el. ~ne vol-ume of precipitate was about 2% of the initial broth volume.
The precipitate was collected in a vessel~samples were ~iLh-drawn and it was controlled that all bacteria had been'dlled.
The procedure described in this Example was carried out under aseptic conditions~ i.e. the solutions added and the equipment used had been sterilized before coming into contact with the fermentation broth.

.
.. . .
.

Example 5 The precipitate collected as described in Example 4 was used for the production of a H. pleuropneumoniaevaccine.
A vaccine ~Jas obtained by diluting the preci~itate wi~h phosphate buffered saline (physiological) (PBS) solution of the composition shown in Table V to about the desired del~ity of the vaccine. Then "Thiomersal" [sodium 2-(ethylmercuri-thio)benzoate] and optionally Alhydrogel were added and the precipitate, PBS solution, "Thiomersal" and Alhydrcgel ~ere suspended in a sodium chloride-~hosphate buffer having a pH
of 7, stirred for 20 minutes and a sample withdrawn for ana-lysis. On the basis of this analysis, the densityof tne vac-cine was adjusted to the desired value. Then a Bordetella pertussis suspension (a B. pertussis vaccine) was added and stirrin~ continued for 30 minutes. The density w~ controlled and the vaccine was filled into capped vials.
It will be understood that all of the steps de-scribed in this Example were performed under aseptic con- i ditions.
For subsequent use in a vaccination of pigs, it has been found that a suitable density of the vaccine is about 20 I.O.U. and conse~uently it is preferred that the precipitate of Example 4 is diluted with PBS solution to about this value and that the final density of the vaccine i5 adjusted to a level of 20 I.O.U. be''ore the addition of the B. pertussis susnension. lt has also been found that a suitable co~centration of the adjuvant "Thiomersal" is about 0.01% w/v and that the adjuvant Alhydrogel should preferably be present in a concentration of about 3% v/v.
When adding Alhydrogel it should be taken into consideration that at least the main part of this adjuvant has already - been added during the working-up of the fermentation broth described in Example 4. This involves that generally only a minor adjustment of the content of Alhydrogel is necessary and it may even be superfluous to add Alhydrogel together with the "Thiomersal".

3 2 11~9'^f90 The Bordetella Pertussis suspension which if ~esired may also be added in the form of B. pertussis biomass andJor extracts thereof, is the ingredient in the~I. pleuropneumonia vaccine of this invention which results in the surprisingly superior properties and activities of the vaccine of the in-vention when used for vaccination of pigs agail~t pleuropneu-monia, cfr. the ~linical test results re~orted below.
Neither the B. pertussis vaccine ~er se (biomass and/or extracts) nor the method for its production form part of the present invention which involves that any such vaccme is utilizable as adjuvant in the H. pleuropneumoniae vaccine of the invention, provided that the B. pertussis ~accine as well as the final vaccine of this invention m~et all require- !
ments set out for vaccines according to national andfor inter-national standards. Thus, the preferred B. pertussis vaccine - for use in the production of a H. Dleuropn~umoniae vaccine OL
this invention is a Vaccinum pertussis which nas ~een prepared, ; identified and tested as described in the European Pharma-copoei.a, Vol. III (1975), 409, and complies with the tests mentioned therein.
A suitable amount of the adjuvant B. pertussis suspension in the H. pleuropneumoniae vaccine of the inven-tion is about 16 I.O.U. vaccine which may De con~el~ly ~b-tained by the addition of 100 mls. B. pertussis suspension 2S having a density of 160 I.O.U. to 1 liter of suspension ob-tained by mixing the precipitate of Example 4, PBS solution, "Thiomersal", ~lhydrogel when used, and sodium ~llorid~-phos-phate buffer.
The various densities were measured on a "Corning Colorimeter Z52" at a wave length of 600 nm in the manner described above.
The vaccine is transferred to capped vials in any convenient conventional way.
I' 79(:~

Example 6 By following the procedure described in Example 5, a H. pleuropneumoniae vaccine comprising H. pleuropneumoniae bacteria lO9 cells Alhydrogel 0.03 ml.
"Thiomersal" O.l mg.
Vaccinum pertussis Ph. Eur. 1160 I.O.U.) O~l ml.
Sodium chloride-phosphate buffer (pH 7) to l ml.

was prepared.

l~ Clinical Investigations The vaccine of this invention has been found to be effective in combating pleuropneumonia in pigs exposed ; t~;infection by the microorganism Haemophilus pleuropneu-moniae and it is thus indicated for the prophylactic treat ment of pigs, in particular seronegative pigs, before they are moved to an environment which may already be infected by ~. pleuropneumoniae or where a risk of such an inection may exist. In order to combat pleuropneumonia, the ~igs are 2S vaccinated with a H. pleuropneumoniae vaccine of this inven-tion by intramuscular or subcutaneous injection of the vac-cine, subcutaneous injection being preferable.
In order to ensure an effective vaccination, the vaccine of this invention is administered twice with a suit-able p~riod between the Lnjecticns, preferably not less than 3 we~s.The dosage administered will of course depend upon the body weight of the pigs, the potency of the vaccine, the risks of side effects and the nature of such ef~ects when present, and other factors commonly taken into consideration when effecting a vaccination, irrespective of the active substance in the vaccine. However, a dosage of 2 mls. of the vaccine .
I

described in Example 6 has been found to be effective in general in pigs having a body weight of up to 30 kgs. For pigs of a body weight of above 30 kgs., a dosage of 4 mls~
of the vaccine is suitable.
The following Examples show the advantages obtained by using the vaccine of this invention for vaccination ag ~ st pleuropneumonia compared to prior art vaccines com~rising other adjuvants.

Example 7 45 pigs weighing about 25 kgs. were given a su~cu-taneous injection of the vaccines A to E described in more - detail below. 3 weeks latex the vaccination was re~eated.
The dosag~ was 2 mls., 5 mls. and 6 mls., res~ectively.
3 weeks after the second injection the pigs were challenged with H. pleuropneumoniae bacteria (appr~tely 101 H. pleu pneumoniae bac-teria per animal) and 3 pigs which had receive~
no vaccination were also challenged and use~ as controls. The control a~imals died, and the vaccinated animals survived.
After slaughtering, the animals were examined in order to establish whether the infection had resulted in changes of the lungs and/or changes which might affect the value of the slaughtered animal as being fit for human consumption.
The vaccines used in this test vaccination were the following:

A. H. ~leuropneumoniae cells were cultivated on agar plates and harvested by a PBS solution (composi-tion, cfr. Table V) containing a 0.01% w/v solution of t'Thiomersal". The cell sus~ension containing about 101 cells per ml. was ex~racted for 30 minutes at 56C. and then centrifuged and 1 part by volume of Freund's Incom~lete Adjuvant was added to 3 p~
by volume of suPernatant. Dosage 2 mls. (twice~.

.

., . As vaccine A except that the extracted cells were boiled at 100C. for 2 hours before centrifugation.
Dosage 2 mls. (twice).

5 C. The cells were cultivated as vaccine A, harvested by P~S solution containing a 0.01% w/v solution of "~hiomersal" and adjusted to a density of 20 I.O.U. and then 50~ of Freund's Incom~lete Adjuvant was added. Dosage 5 mls. (twice) con-taining about 109 H. pleuropneumoniae cells ~er ml.

D. H. pleuropneumoniae cells were cultivated in a fl~k until the density was 5 I.O.U., killed by for~alde-hyde and precipitaied by the addition of Alhydrogel 1~ as described in Example 4. The precipitate was diluted with PBS solution to a concentration o 1 mg/ml. having a content of about 109 H. pleuro-pneumoniae cells per ml. Dosage 5 mls. (twice).

20 E. A vaccine of this invention of the composition ~iven in Example 6 and prepared substantially as oescribed in Examples 2, 4 and 5. Dosage 6 mls. ~twlce).

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

3a , Table IX

Vaccme Nu~ of Clini~ Changes R~ks by H. p.
used ~ slau~h~rin~ isolated A 10 2/10 ) 0/lOa) l/lOa) N.D. ) B 10 4/10 1/10 0/10 N.D.
C 11 1/11 2/11 1/11 2~2 E 6 0/6 0/6 0/6 W.D.
None 3 3/3 ) - 3/3 3/3 ~Con-- trol~
a~ Number of animals with symptoms tchanges, remarks)/nu~ber of test animals.
b~ Not Determined.
~) 1 animal died, 2 ~ere killed when dying~

From Table IX, it will be seen that vaccine E o~
this invention is superior to the prior art vaccines A to D
in providing protection against infections caused ~y ~. pleurcr pneu~oniae bacteria~ Vaccine E is the only one in whi~h nei~her symp~oms nor changes of lungs and/or remarks by slaughtering are observed. Changes of the lungs,viz. scars in the ung tissue as a result of a pleuropneumonia attack, were cbserved in 8 uf the 45 vaccinated animals. By slaughtering chronical pleurisy was remarked in 4 of the animals. However~ all of the 45 animals were acceptable for human consumption~

~ E:~amp]e 8 A further test vaccination was carried out on 46 pigs and 2 animals were used for control purposes~ The animals ~eighed about 25 kgs. The vaccinations and challenge infec tions were carried out in the manner described in Example 7 The ~osages used for vaccination were 2 times 2 mls.

;

3'7~9 The primary purpose of this test is to compare the effect of the Bordetella pertussis adjuvant of this inven-tion to the effect of various other adjuvants of which some have already been used as such adjuvants in the prior art.
The control animals survived until slau~htering, but 2 of the vaccinated animals died before slaughtering.
One pig of group L was killed and clearly showed signs of pleuropneumonia. One pig of group E died due to fighting, i.e. ~or a reason without connection to the present test.
Examination of the lungs revealed no signs of pleuropneu-monia. The slaughtered animals were examined for the pur-poses described in Example 7.
The compositions of the test vaccines are given below. It should be noted that "vaccine" L is no vaccine in the common sense of this term, however, the results ob-tained demonstrate that pleuropneumonia was not success-fully controlled by pernasal and peroral adminis~ration of the active ingredient in the vaccine of this invention.
The vaccines used were the following:

C, D, E. As in Example 7.
F. A vaccine of the composition of Example 6 (vac-cine E), except for a content of "Quil A" as the adjuvant instead of Vaccinum pertussis. "Quil A"
is a saponine derivative of known composition which has been disclosed by K. Dalsgaard in Acta Vet.
Scand., Suppl. 69 (1978).

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

~. As F, except for a content of "Levoripercol" as the adiuvaht instead of "Quil A"~ "Levoripercol" is Levamisolum NFN anthelminticum sold by the firm 31 ~-. fi8~

3~ 9~

Lundbeck, Copenhagen, Denmark. Levamisolum is a Non-Proprietary Name approved by NFN (The Nordic Pharmacopoeia Council).

I. As vaccine E, except for a content of "Lymphocyte Proliferative Factor" (LPF) as the adjuvant instead of Vaccinum pertussis. The LPD was e~tracted from Bordetella pertussis bacteria as disclosed by S.I. Morse and J.H. Morse, cfr. J. Exp. Med. 143 10 (1976), 1483-1502.

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

L. H. pleuropneumoniae cells were cultivated on an agar plate, harvested and transferred into an aerosol which were atomized into the nose and mouth of the animals.
Of the vaccines listed above, vaccines E, I and K
are within the scope of the present invention.
The resul-ts of the test vaccination of this Example are summarized in Table X.

Table X
Vaccine Num~er of Clinical Changes ~rks by H. p.
used _ test nimals s~mptoms of lungs slaughtering isolated 3 0/3 0/3a) 0/3a) NoD~b) D 5 0/5 0/5 0/5 N.D.
E 10 0/10 0/10 1/9 ) N.D.
30 F 10 0/10 0/10 0/10 N.D.
G 2 0/2 1/2 1/2 N.D.
H 5 0/5 0/5 1/5 N.D.

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

~I nr 686 37~3~
3g Table X (continued) Vaccine Number of Cl ~ cal Changes R~marks by H~ p.
used test animals s~m~to~~s _o lunas slaughterin~ lsolâted 5 I 5 0/5a) o/5a~ o/5a) N.D,b) K 4 0/4 1/4 n/4 N.D.
L 2 2/2 2/2 l/lc) 1/2 None 2 2/2 2/2 2/2 N.D.
(Co~--~1) " i a) Num~er of animals with symptoms (changes, remarks)/n~mber of test animals~
b) Not Determined.
c) 1 animal died before slaughtering.
From Table X it will be seen that clinical sympto~s - only occurred in aerosol-treated animals and in the control animals. Of the remaining 44 animals, only 2 showed changes o~ the lungs, and only 3 gave rise to remarks a-t slaughteriny although it could be demonstrated by serological examinations that all an~mals had been attacked by ~. pleuropneumonia~.
Thus, ~able X shows that vaccination with a H. pleuro--pneumoniae vaccine comprising killed H. pleuropneumoniae bac~
teria prepared along the lines disclosed in Examples 2, ~, and 5 above as the active ingredient is very effective for protecting pigs against pleuropnellmonia attack. :'-ltho~h the various adjuvants tested may seem equally well suited accord-ing to Table X, the B. pertussis vaccine adjuvant (or bio~
mass and/or extracts thereof) used according to the invention ~ I
30 is superior and thus preferred because it results in an opti- ¦
mal pro~ection and no or only a sligh, and short reaction on the injection site whereas the prior art adjuvan-ts tend to give rise to granuloms on the injection site.
It will be understood that the above description and 3S Examples are only to be considered as being embodiments o~ the inventi~ a~d it will be bvi~s to those sl:illed in the art that ~

¢~
o fications and variations may be effected therein without departing from the spirit and scope of the invention as defined in the appended claims. In particular, it will be understood that the invention is not restricted to the use of the specifically mentioned strain and/or subculture of H. pleuropneumoniae. Thus, also cultures obtained from said strain and/or subculture by natural and/or artificial selection and/or mutation as well as cultures of any of - the various serotypes are within the scope of the present invention.

' . , 3~

35 .

I nr ~5

Claims (6)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

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

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

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

4. A vaccine according to any of the claims 1 to 3, 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.

5. A vaccine according to any of the claims 1 to 3, characterized by a content of about 109 cells of H. pleuro-pneumoniae per ml.

6. A vaccine according to any of the claims 1 to 3, characterized by having the composition:
H. pleuropneumoniae bacteria 109 cells Alhydrogel 0.03 ml "Thiomersal" 0.1 mg Vaccinum pertussis,Ph. Eur. (160 I.O.U.) 0.1 ml Sodium chloride-phosphate buffer (pH 7) to 1 ml.