DE3517805A1 - Vaccine for immunising pigs against atrophic rhinitis - Google Patents

Abstract

The present invention relates to a vaccine for immunising pigs against atrophic rhinitis, based on the bacterial strain Bordetella bronchiseptica CNCM No. 1436 and Pasteurella multocida CNCM No. 1437, a process for its production and its use.

Description

Vaccine for the immunization of pigs against rhinitis

atrophicans (sniffing disease) The present invention relates to a new vaccine to immunize pigs against atrophic rhinitis (RA) based on microorganisms of the genus Bordetella and Pasteurella, method for its manufacture and use.

Atrophic rhinitis (sniffing disease) affects pigs especially piglets and runners. The disease occurs and calls in almost every country significant due to deaths, delayed growth and poor feed conversion economic damage.

No specific RA pathogen has yet been found. Yet is now widely believed to be an infectious primary cause. During the examination Diseased or slaughtered animals were particularly often infected with Pasteurella multocida, Bordetella bronchiseptica, Haemophilus suis, Pseudomonas aeroginosa and Mycoplasma hyorhinis found.

Atrophic rhinitis is mostly caused by droplet infection of pigs transferred to pig.

Prophylactic and therapeutic administration of antibiotics and sulfonamides have proven themselves in the fight against atrophic rhinitis.

However, they require ongoing treatment of the animals with medication or medicated feed, it is known to immunize against atrophic rhinitis to use vaccines inactivated by formal in, ultrasound or thimerosal, with germ concentrations between 1 - 6 x 1012 germs / ml. The vaccines are simple- or combination vaccines (Bordetella / Pasteurella), the most diverse are described Sows and piglets vaccination programs. The dosages also vary between 0.5 ml and 20 ml per injection (see US-P 4 203 970>.

There are already combination vaccines with inactivated Bordetella and Pasteurella strains against RA on the market, they contain as adjuvants either Aluminum hydroxide or oil adjuvants of unknown composition, The immunoprophylaxis against rhinitis atrophicans is not yet satisfactory in practice, so that there is considerable economic interest in the provision of further vaccines It has now been found that an excellent vaccine against atrophic rhinitis based on the trunk Bordetella bronchiseptica with the CNCM No. 1 436 and Pasteurella multocida with the CNCM No. I 437 as well as their variants and mutants can be produced.

There was also a new vaccine against atrophic rhinitis the base of the Bordetella bronchiseptica strains with the CNCM No. I 436 and Pasteurella multocida with the CNCM No. I 437 as well as their variants and mutants found Es also the strains Bordetella bronchiseptica with the CNCM No. I 436 and Pasteurella multocida found with CNCM No. I 437.

The strain Bordetella bronchiseptica (red) was on April 24, 1985 under of the CNCM No. I 436 at the Collection Nationale de Cultures de Microorganismes (Institut Pasteur) 25, rue de Docteur Roux, Paris.

The strain Pasteurella multocida (R2) was registered on April 24, 1985 under the CNCM No. 1 437 at the Collection Nationale de Cultures de Microorganismes (Institut Pasteur) 25, rue de Docteur Roux, Paris.

Strain R1 (Bordetella bronchiseptica) strain R2 (Pasteurella Multocida was. mis) The R1 strain was created in Spain in 1978 from rabbits that had come into contact with RA patients Pigs were isolated.

The R2 strain was isolated from a pig in 1975.

Identification by immunofluorescence with specific antiserum the ATCC, Identification using the API 20E test system of Company API System SA La Balme les Grottes 38 390 Montalien Vezien, France. Thereafter the following biochemical reactions are carried out and the following results are obtained: R1 R2 (Bordetella) (Pasteurella) ONPG CIT + IND - + MAN - + SAC OX + ADH SH2 VP + INO MEL NO2 + + LDC HREA + Gel SOR - + AMY ODC TDA GLU - + RHA ARA - + Both strains are gram-negative.

They grow on brain-heart-infusion agar (company Difco) with typical colony shape.

Bordetella grows in smooth, raised, shiny colonies, Pasteurella grows in the form of round, small (diameter 1-3 mm) gray colonies, both stems can be used in the usual way, e.g. as lyophilisate or in slanted tube agar or in liquid nitrogen.

The strains are propagated in the usual way in nutrient media which contain the usual carbon and nitrogen sources and the necessary salts under aerobic conditions.

The following nutrient media may be mentioned: brain-heart-dextrose broth, brain-heart-infusion, or brain-heart dextrose broth agar, brain-heart infusion agar.

For example, brain-heart dextrose broth has the following composition per liter: Calf brain infusion 12.5 g Bovine heart infusion 5.0 g Proteose-Tepton (Oxoid L 46) 10.0 g dextrose 2.0 g common salt 5.0 g disodium phosphate 2.5 g pH = 7.4 Brain Heart Dextrose Broth Agar has the following composition per liter, for example: Calf brain infusion 12.5 g beef heart infusionla 5.0 g proteose tepton (oxoid L 46) 10.0 g dextrose 2.0 g common salt 5.0 g disodium phosphate 2.5 g agar No. 1 (Oxid L 11) 10.0 g pH = 7.4 As carbon sources can be used are: carbohydrates, especially polysaccharides, such as

Starch, monosaccharides such as glucose or fructose.

Sugar alcohols such as mannitol can also be used or glycerine, as well as naturally occurring mixtures such as malt extract or distiller soluble and mixtures from the sources mentioned.

The usual nitrogen sources can be used as nitrogen sources such as proteins, protein hydrolysates, amino acids, ammonium salts, complexes Natural products such as soybean meal, meat extracts, yeast extracts, milk powder and suitable mixtures thereof.

Mineral salts are required as auxiliary substances in the nutrient medium, e.g. phosphates, Sulfates or chlorides, of potassium, sodium, calcium, magnesium, Iron, zinc and manganese. The concentration of these substances can be within wide limits fluctuate, in some cases the necessary concentrations of the mineral salts in the above

Carbon or nitrogen sources or in water used as Contain impurities.

Furthermore, antifoam agents of the most varied can also be used as auxiliaries Kind of use, such as

Polyols or silicones.

The trunks can be solid, semi-solid or liquid with the help of usual Culture media are propagated. Aqueous liquid nutrient media are preferred.

The inoculation of the nutrient media takes place in accordance with the generally customary methods Methods, e.g. using slanted tubes or flask cultures.

The culture takes place under aerobic conditions and can according to the general common methods such as using shake cultures e.g. in shake flasks, from air-moving cultures or from submerged cultures. Preferred the cultivation takes place in aerobic submerged processes in aerated fermenters, e.g. in common submerged fermentation tanks. It is possible to keep the cultures continuously or to be carried out discontinuously. The process is preferably carried out batchwise.

The percentage ratios of the nutrient solution components can be wide Ranges vary, generally the carbon sources make up 0.5 - 8%, above sometimes 0.5-6%, the nitrogen sources 0.1-5%, preferably 0.5-2%, the salts are present in the usual concentrations, preferably in the range between 0.001-2% by weight. The antifoam agents are present in about 0.5% concentration.

The pH of the growing cultures should preferably be between about 5 and about 10, in particular between 6.5 and 8.0. Too strong pH drop in the acidic range can be caused by additions of an organic or inorganic Base, preferably CaCO 3, should be avoided. As in fermentation technology Usually, an automatic pH regulation can also be carried out during the sterile organic or inorganic acids, e.g. H2S04, or sterile alkalis, e.g. NaOH can be injected into the culture solution at intervals. Or the pH will controlled by blowing in sterile C02-containing air.

It is useful to ensure that the microorganisms are sufficient be brought into contact with oxygen as well as the nutrients. This can be done after the generally customary methods such as shaking or stirring.

The growth temperature can be between about 15 ° C and about 50 ° C, preferably between 20 and 40 "C, it is particularly preferably around 37" C. The duration the cultivation can be varied widely, e.g. the composition of the nutrient medium and the growth temperature play a role. The respective optimal conditions can easily be determined by anyone skilled in the microbiological art.

As is generally the case with microbiological processes, foreign infections of the culture media are avoided.

The usual precautions are taken for this, such as sterilization the culture media, the culture vessels and the air required for ventilation. Steam as well as dry sterilization can be used to sterilize the device be used. The temperatures used for sterilization are around 100 - 200 "C, preferably at 120-1300C.

If undesirable amounts of foam are produced during cultivation, the usual chemical foam suppressants, e.g. liquid fats and oils, Oil-water emulsions, paraffins, higher alcohols such as octadecanol, silicone oils, polyoxyethylene or polyoxypropylene compounds are added. Foam can also be used with the conventional mechanical devices (which e.g. use centrifugal forces) are damped or be eliminated.

The cultures obtained in this way can either be used directly for production of the vaccine can be used, or the cells of the culture in the usual Way (e.g.

by centrifugation or filtration) separated and further in the usual way Formulated wisely.

To produce the vaccine, the bacterial strains are either inactivated before or after their union. Inactivation takes place through the usual Inactivating agents such as formalin, beta-propioactone, hydroxylamine, ethylene oxide, Ethylenimine and its derivatives, chloroform, phenol, thimerosal. Formal is preferred in.

Is the inactivation e.g. with formalin before the cells are separated If necessary, a special neutralization can be carried out by separating the cells of the inactivating agent can be avoided.

Inactivation with formal in (37-40%) takes place at formalin concentrations from 0.1-2 percent by volume, preferably 0.5-1 percent by volume.

For inactivation with Formalin, culture for 6-100 hours is preferred 48-84 hours, particularly preferably 70-80 hours, treated at 37.degree.

Inactivation can also be done with thimerosal (sodium ethyl mercurithiosalicylate) can be carried out in a concentration of approx. 1: 104.

The inactivated microorganisms can be mixed with suitable adjuvants to increase the effectiveness.

Such adjuvants are e.g. complete or incomplete Freund's Adjuvant, aluminum oxides, hydroxides, oxigels, salts such as phosphates, stearates as well as alginates such as sodium alginate. Sodium alginate and calcium gluconate are particularly preferred.

Non-oily adjuvants are usually in concentrations in the vaccine from 1-10 percent by weight, preferably 5-10 percent by weight. Oily adjuvants are usually present in the vaccine in concentrations of 40-60 percent by volume.

The vaccines can contain other additives such as preservatives or contain buffer substances. Formalin as well as serve as preservatives Thimerosal.

Phosphates such as disodium hydrogen phosphate serve as buffer substances. Carbonates such as sodium hydrogen carbonate.

In the vaccines, the inactivated bacterial strains are in one Salary from 5 - 30. 109 germs per ml. Preferred are 15. 109 germs per ml. The ratio of the content of both bacterial strains is about 1: 1.

The vaccines have a pH of 6-8. A pH is preferred from 7.

The vaccines produced by the process of the invention are used to vaccinate pigs.

Pregnant dams are preferred, but also all piglets Vaccinated ages.

Vaccination is carried out parenterally by injection (intramuscular or subcutaneously) or by spraying the mucous membranes of the nose and throat with the vaccine.

The vaccine is used for vaccination in quantities of 2-3 ml per animal.

Vaccination is preferred after 3-4 weeks and then every 3-4 months repeatedly to ensure lasting protection.

Example 1 With 20 ml of an inoculum from Brain Heart Infusion in a sample of the Bordetella CNCM No. I 436 strain was cultivated at 37 ° C. for 24 hours 20 l sterile nutrient solution of the following composition are inoculated: Na2H P04 244 g NaCl 11 g Mg S04. 7 H20 2g glucose 100 g yeast 80 g water ad 20 1 die Culture was kept at 37 ° C. for 24-45 hours and shaken 2-3 times a day.

Example 2 The procedure given in Example 1 was used with the strain Pasteurella CNCM No. 1 437 repeated.

Example 3 The cultures obtained from Examples 1 and 2 were then so much formal in (37-40 X) was added that the formalin concentration was 0.8 The inactivation of the bacterial strains occurred during 72-96 Hours at 37WC with stirring EXAMPLE 4 Those obtained according to Example 3 Cultures of inactivated bacteria were then centrifuged and the cell material absorbed in so much physiological saline solution (0.9%) that a content of 1012 germs per ml arose. The resulting solutions of both strains were then combined in the ratio 1: 1 and with 6. 10 4 percent by weight thimerosal added.

Example 5 The solution of both strains from Example 4 was with so much sterile 15% aqueous sodium alginate solution and so much sterile 20% aqueous Calcium gluconate solution added that the final concentration of sodium alginate and the The final concentration of calcium gluconate is 4% in each case. The pH of the solution was adjusted to 7.2 by adding 20% aqueous NaOH. The whole solution was kept at 37 ° C. for 24 hours and then filtered off under sterile conditions.

Example A 2 pregnant pigs are 7 weeks and 2 weeks before the Throw subcutaneously with 2 ml vaccines according to the invention (prepared according to Example 5) each time The piglets born were vaccinated at the age of 16 days with a field strain of Bordetella brochiseptica infected intranasally.

Piglets of the same age from unvaccinated pigs were used as controls The piglets of the vaccinated pigs showed infections after 3-6 days post no fever while the piglets of the unvaccinated pigs had a temperature increased by 10C showed after the end of the experiment (approx. 18 days after infection) the mortality certainly. In the protected pigs, none of the 16 piglets died, O X the piglets had RA (pathologically anatomically determined). The control animals died 3 of 18 piglets, 88 X of the piglets had RA (pathologically anatomically determined).

Claims (5)

Claims 1. Process for the preparation of a vaccine against Atrophic rhinitis (sniffing disease in pigs), characterized by that for the production of the vaccines the bacterial strains Bordetella bronchinseptica with the CNCM No. I 436 and Pasteurella multocida with the CNCM No. I 437 as well as the Variants and mutants of these strains are used. 2. Vaccines against rhinitis atrophicans (snuffle disease in pigs), characterized by a content of a mixture of the bacterial strains Bordetella bronchiseptica CNCM No. I 436 and Pasteurella multocida CNCM No. I 437 and their Variants and Mutants. 3. Bacterial strain Bordetella bronchinseptica CNCM No. 1 436 and its variants and mutants. 4. Bacterial strain Pasteurella multicida CNCM No. I 437 and its variants and mutants. 5. Use of vaccines according to claim 2 for the immunization of Pigs against atrophic rhinitis.