GB2176705A - Live vaccine effective against sheep and goat listeriosis - Google Patents

Abstract

Attenuated strains of Listeriosis monocitogenes deposited in "NBPMKK" under numbers 251 and 252 are cultivated on meat-peptone bouillon in a fermenter, the bouillon containing 8-20 g/l glucose. Cultivation is at 35-37 DEG C and pH from 7 to 7.6, with air passed through preferably at 3-5 l/min, and a stirrer operated at 300 to 1000 r.p.m. The amount of innoculum is from 1:20 to 1:100 of the working volume of the fermenter. After termination of the cultivation, depending on the density of the biomass obtained, the latter is diluted with protective medium and then it may be homogenised and lyophilised. A biomass having a density of 12 milliard/ cm<3> living bacterial cells may be obtained. For lyophilising of the vaccine, a protective medium containing 2 to 17% by weight sodium glutamate, 5 to 20% saccharose and 0.5 to 5% by weight denatured gelatine is presently used.

Description

SPECIFICATION Method for the preparation of live vaccine effective against sheep and goat listeriosis This invention relates to a method for the preparation of a live vaccine effective against sheep and goat listeriosis.

A method for the preparation of sheep and goat listeriosis vaccine is described by I. Ivanov in "Study of the active immunopropylaxis of sheep listeriosis", Proceedings of the Vllth International Symposium on problems of Listeriosis, 1979. In this method the strains Listeria monocitogenes 1/2a No. 326 and 4B No. 125 deposited in "PBPMKK" under Nos. 251 and 252 respectively are statically cultivated in meat-peptone bouillon or meatpeptone agar. The biomass obtained from both strains is used to produce native or lyophilised vaccine.

This method for the preparation of the vaccine has the following disadvantages. The bacterial suspension obtained in the static cultivating of the bouillon has a low density which makes it difficult to prepare a lyophilised vaccine. Production of cultures on agar is labour intensive with a high percentage of rejects due to contamination with other bacteria.

There is thus no practical possibility of producing the vaccine industrially. Furthermore, with static cultivating, many of the bacterial cells perish during the cultivation. After lyophilising, the survival rate for listeriosis cells amounts to only 30 to 50%.

According to the present invention, there is provided a method for the preparation of a live bivaccine effective against listeriosis in sheep and goats, which comprises cultivating together attenuated Listeria monocitogenes 1/2a and 4B strains deposited in "NBPMKK" under the Nos. 251 and 252 respectively, on meat-peptone bouillon containing 8 to 20 gll sucrose, cultivation being carried out in a fermenter through which air is passed and in which stirring takes place, the cultivation temperature and pH being from 35 to 37"C and from 7 to 7.6 respectively, with the innoculum occupying 120th to 100th of the working volume of the fermenter, terminating cultivation and optionally diluting the biomass obtained with a protective medium.After ending the cultivation, depending on the density of the biomass obtained, it is used as such or it may be diluted with a protective medium and again used as such or homogenised and lyophilised to be reconstituted subsequently for use.

The fermentation cultivating is carried out as follows: the necessary innoculum is obtained from vaccine strains Listeria monocitogenes 1/2a and 4B and is introduced into the fermenter. A meatpeptone bouillon with 8 to 20 g/l glucose is lyophilisation is subsequently to take place contair 2 to 70% by weight sodium glutamate, 5 to 20% by weight saccharose and 0.5 to 5% by weight denatured gelatine.

During the lyophilisation process, the temperature of the product during the period of sublimation of water is maintained below -30"C.

After ending this process, the temperature is increased to 40 C. The vacuum in the chamber is from 0.1 to 0.05 mm/Hg. The temperature of the condenser is below 45"C. After termination of drying, the vacuum is released by admitting a neutral gas which fills the vaccine containers.

When preparing live bi-vaccine effective against sheep and goat listeriosis by the method of the invention, a biomass is produced which is uniform possesses high density, contains a high percentagi of live cells free from the risk of contamination by secondary microflora and which preserves the initi properties of the vaccine's attenuated strains after lyophilising. Moreover, the method according to tP invention can be automatised. The vaccine obtaine shows a survival rate for the cells of both serotypez which is not lower than 80% and has been found tc have a period of validity of as much as one year.

Hence a product is produced which is stable and which results in high live cell content, immunogen characteristics, harmlessness and purity, as well ac containing the two strains used in equal quantities The method thus developed permits the manufacture of vaccine on industrial scale.

The following Examples illustrate the invention: EXAMPLE 1 Fermentation Cultivation of Strains 1/2a and 4B of L.monocitogenes Lyophiiised samples containing vaccine strains were suspended and cultivated in a meat-peptone bouillon with 1% by weight glucose. The ampoule! were incubated for 24 hours and then sub-cultures were prepared and introduced into the fermenter ii an amount of 1/20 of the working volume of the fermenter which was 7 litres. The cultivating was effected under the following conditions: air fed through at the rate of 3 to 5 I/min; stirrer operated 300 rpm; temperature 37"C and pH 7.2.

At every stage of the operation, the purity was checked by means of a preparation coloured after "Gram" into which samples were innoculated, cultivation occurring in Petri dishes with meatpeptone bouillon.

At the end of the fermentation cultivating, all reproduction was interrupted by having deceleration of the rate of multipiication take place A biomass with a density of up to 12 milliard/cm3 living bacterial cells was obtained. The density of the bacterial suspension of sub-culture and determined by the density of the biomass obtained in which the ratio of the two vaccine strengths was 1:1. After homogenising the biomass with the protective medium, the vaccine thereby produced was poured into bottles (vials) or metal containers suited to the needs of the consumer.

EXAMPLE 2 Lyophilisation of a Vaccine The vaccine produced in the preceding Example was frozen in advance in a static or rotary freezer depending on the form of packaging containing the vaccine. The vaccine was then hydrophilised in a iyophilising chamber. During the process, the temperature of the product during the period of sublimation was maintained below -30 C. After termination of this process, the temperature was increased to 40"C. The vaccine in the chamber was 0.1 to 0.05 mm/Hg. The temperature in the condenser was below -45 C. After termination of the drying, the vacuum was broken by admission of a neutral gas (nitrogen) with which the containers were filled.The product obtained was stored in a dry, dark place at a temperature of 4 to 8"C. Make-up solvent used for reconstituting the vaccine was a physiological solution containing 0.1 g/l saponin and of pH 7.2. After reconstituting the vaccine by placing the lyophilisation product in suspension, the vaccine contained not less than 1 milliard/cm3 living bacterial cells in total belonging to both serotypes in approximately equal quantities.

After the lyophilisation, the vaccine was checked forthe number of living bacterial cells by cultivation thereof on innoculation into Petri dishes. Safety with guinea pigs and immunogenic properties were determined. No conjunctivitis appeared on conjunctival treatment of guinea pigs according to the method of Anton, in which there is initial dropping of vaccine into the eyes with administration after at least 12 days in like manner of cultures from the pathogenic strains L.monocitogenes 1/2a and 4B. In contrast to the controls not treated initially with the vaccine, there was a clearly expressed conjunctivitis with pus formation. No conjunctivitis was observed with animals previously given the vaccine in the aforesaid manner.

EXAMPLE 3 The vaccine of each of Examples 1 and 2 was applied once to a sheep, a goat and to a lamb and a kid aged over 6 weeks in a dose of 2 ml. The animals were injected hypodermically behind the elbow joint. Immunity was obtained 12 days after the vaccination and lasted for 10 months.

Claims (7)

1. A method for the preparation of a live bivaccine effective against listeriosis in sheep and goats, which comprises cultivating together attenuated Listeria monocitogenes 1/2a and 4B strains, deposited in "NBPMKK" under the numbers 251 and 252 respectively, on meat-peptone bouillon containing S20 g/l sucrose, cultivating being carried out in a fermenter through which air is passed and in which stirring takes place, the cultivation temperature and pH being from 35--37"C and from 7 to 7.6 respectively with the innoculum occupying 1/20 to 1/100 of the working volume of the fermenter, terminating cultivation and optionally diluting the biomass obtained with a protective medium.

2. A method as claimed in claim 1, wherein, during cultivation, air is passed at a rate of 3 to 5 I/minute.

3. A method as claimed in claim 1 or 2, wherein stirring of the bouillon takes place at 300 to 1000 r.p.m. during cultivation.

4. A method as claimed in any preceding claim, wherein the biomass obtained at termination of cultivation, is homogenised and then lyophilised.

5. A method as claimed in any preceding claim, wherein the protective medium contains from 2 to 17% by weight sodium glutamate, 5 to 20% by weight saccharose and from 0.5 to 5% by weight denatured gelatine.

6. A method for the production of a live bi-vaccine effective against listeriosis in sheep and goats, substantially as described in either of Examples 1 and 2.

7. A live bi-vaccine effective against listeriosis, whenever produced by the process claimed in any preceding claim.