DD267864A7 - METHOD FOR THE PRODUCTION OF A LIVING VACCINE AGAINST LISTERIOSIS IN SHEEP AND GOATS - Google Patents

Abstract

In the proposed method of producing a live vaccine against listeriosis in sheep and goats, attenuated strains of the species Listeria monocitogenes are cultured in a meat-peptone broth containing 8 to 20 g / l glucose. The culture of the strain is carried out in a fermenter while maintaining the following characteristics: air from 3 to 5 l / min., Mixer from 300 to 1,000 revolutions / min, temperature of 35 to 37C, p H value of 7 to 7.6 , the quantum of Innokolums is from 1:20 to 1: 100 per working content of the fermenter. After completion of the cultivation, the biomass produced is diluted, depending on its density, with a protective medium, then homogenized and lyophilized. The lyophilization is carried out in a protective medium having a composition of 2 to 17% sodium glutamate, 5 to 20% sucrose and 0.5 to 5% of destructed gelatin. The applied lyophilization method of the vaccine ensures no less than 80% surviving cells of both serotypes.

Description

Ancestors for the production of a live vaccine against the colony of sheep and goats

The invention relates to a method for producing a live vaccine against listeriosis in the f: hafon and Ziogon as Prävenjivmlttel, which will find application in veterinary medicine.

There is known a process for the production of a vaccine against listeriosis in sheep and goats, according to which the strains Listeria monocitogenes 1/2 a no. 326 and 4 b no. 125 are used, deposited in the National Bank of industr. Microorganisms u. No. 251,252 cell cultures, which are cultured statically in a fl ask peptone broth or fl ask peptone agar. The biomass obtained from both strains is used for a native or lyophilized vaccine / 1 /. The drawbacks of this process for the production of the vaccine consist in that the bacterial suspension produced in the static culture of the broth has a low density, which makes it difficult to prepare a lyophilised vaccine. Culturing on Ana 'is laborious, with a high percentage of rejects, because of contamination with other bacteria, and does not provide a possibility for industrial production of the vaccine. In addition, in static culturing, part of the bacterial cells enter during the cultivating process. After lyophilization, the surviving listeriosis cell counts from 30% to 50%.

The object of the invention was now, a method for the production of a living Bivakzine gogen listeriosis in the To develop sheep and Ziogen, in which a uniform biomass is produced, which has a grosso density, one

high percentage of living cells, without contamination by a minor flora, the

Initial lifetimes of the attenuated vaccine strains are retained after lyophilization. This object is achieved by a method for the production of a live bivaczine against listeriosis in sheep and Goats are dissolved, according to the biomass by cultivating in a fermenter eggs of two vaccine strains Listeria

monocitogenes is produced. The cultivation of the strains takes place in a fermenter with the following parameters:

Air 3 to S l / min, mixer from 300 to 1000 revolutions / minute, temperature from 35 to 37 ° C, pH from 7 to 7.6, the Quantum of the inoculum (the seed culture) is 1:20 to 1: 100 per volume of fermenter. After completion of cultivation

is, depending on the density of the resulting biomass, the latter diluted with olnorr protective medium, then homogenized and lyophilized.

The process of fermentor culturing is ¹ below. The required inoculum culture is obtained from the vaccine strains (vaccinal strains) Listeria monocitogenes 1 / 2a and 4b

prepared and placed in a fermenter. This is in a meat peptone broth with 8 to 20g / l glucose at one

Temperature of 35 ⁰ C to 37 ⁰ C cultivated. The produced biomass has a density of 12 billion / cm ³ living bacterial cell For the lyophilization of the vaccine is a protective medium having a composition of 2 to 17% Nutriumglutamat, 5 bis

20% sucrose and destructured gelatin of 0.5 to 5%, which is added to the biomass, after which the latter is lyophilized.

During lyophilization, the temperature of the product is maintained at sublimation below -30 ^° C. After this

This process is completed, the temperature is increased up to 40 ° C. The vacuum in the chamber is from 0.1 to 0.05 torr. The

Temperature of the condenser is below 45 ⁰ C. After the drying is finished, the vacuum is replaced by a neutral gas

destroyed, with which the packages are filled.

The advantages of the method according to the invention are the following:

- It is possible to automate the production processes.

- ηίη produced biomass is uniform, without contamination by secondary flora and with a high density.

- Pas applied lyophilizing the vaccine ensures not ν ess than 80% surviving cells of both serotypes. This enables the production of a stable product with respect to living cells, with immunogenic properties, harmlessness and purity, as well as the maintenance of the two strains contained in the vaccine in equal proportions.

The invention will be explained in more detail with reference to the examples given below. Example 1: Cultivation in a Fermen.ator of strain 1/2 «and 4b Listoria monocltogonoe fü. one working content 7I. The lyophilized vials with the vaccine strains were resuspended and a flask containing 1% glucose was added. Peptone broth is inoculated. These are incubated 24 hours, then the subcultures are prepared, which are in the Fermontotor

in a quantity of 1/20 from the working contents of the fermenter. The cultivation is followed by the following

Conditions carried out: air 3 to Bl / Mnn .; Mixer 300 revolutions / minute; Temperature 37'C; pH word - 7.2. At each stage, a test for purity by means of a 'Gram' stained preparation and a vaccination of Petri dishes made with Fleiacn peptone broth. At the end of Ferment.itor cultivation, which at the end of decelerating igsphase the propagation speed

interrupted, one biomass with a density up to 12 billion / cm ³ living bacterial cells. The density of the bacterial suspension of the subcultures and the fermenter biomat is also measured with a "Spekol" based on a dos

5. international optical standards for turbidity generated curve determined.

According to the density of the crop «new biomass, in wolcher the ratio of both vaccinal strains 1: 1 is:, becomes Biomass diluted with a protective medium consisting of 2 to 17% sodium glutamate, 6 to 20% sucrose and 0.5 to 6%

destructured gelatin. After homogenizing the biomass with the protective medium, the vaccine in flasks or

Doses poured depending on the needs of the consumer. Example 2: Lyophilizing the vaccine.

The vaccine is previously frozen static or rotating depending on the packaging. For the drying of the vaccine special chamber apparatuses are used for the Lyophllislerung. During the process, the product temperature is maintained at -30 ⁰ C under sublimation. After completion of this process, the temperature is raised up to 40 * C. The vacuum in the chamber is from 0.1 to 0.05 torr. The temperature of the condenser is below -45'C. After the drying is finished, the vacuum is destroyed by a neutral gas (nitrogen), with which the packages are filled. These are stored in a dry and dark place, at a temperature of 4 ° C-8 ^e C. The solvent used is a physiological solution containing 0.1 g / l of saponin with a pH of 7.2. After resuspension, the vaccine contains no more than 1 billion / cm ^{3 of} live bacterial cells of both serotypes in approximately equal amounts. After lyophilization, the vaccine is tested for:

Number of live bacterial cells by inoculation of Petri dishes:

- harmlessness (to guinea pigs)

- Immunogenic properties.

In the conjunctival treatment of the guinea pigs after the Anton ancestor, no conjunctivitis occurs, as well as at the first instillation into the eyes with bivakzine, as well as boi the second instillation, at least after 12 days, with cultures of pathogenon Listeria monucitogenes1 / 2a and 4b, while the controls, which were originally treated with bivakzine in the eye), were given a distinct pus-conjunctivitis.

Example 3:

The vaccine is applied once to sheep, goats, lambs and kids over 6 weeks of age, in a dose of 2ml. The animals are injected subcutaneously behind the elbow joint. The immunity rode after the 12th day and lasted 10 months.

Documents considered:

1. Ivanov I. Study of the Act've Immunoprophylaxis of Shoep Listeriosi3 Proceedings of the VII 'ntern. Sympooium of

Listeriosis, 1979.

2. OzeboldJ.W.DiCapraHAtoJ.Bact., 95,2153,1968.

Claims (2)

1. Method for producing a live vaccine against d ' . Listerlosis in sheep and goats, according to which attenuated strains of the species Ltateria monocitogenes deposited in the National Bank for industrial microorganisms and cell cultures for strain 1/2 a under no. 251 and for strain 4b under no. 252, in one meat Peptone broth cultured with 8 to 20g / l glucose, characterized in that the cultivation of the strains in a fermenter is carried out in compliance with the following characteristics: air from 3 to 5I / min., Mixing of 300 to 1000 revolutions / min., Temperature of 35 to 37 ⁰ C, pH of 7 to 7.6, the quantum of Innokolums is from 1:? 0 to 1: '00 per working contents of the fermenter; After completion of the cultivation, the produced biomass is, depending on its density, diluted with a protective medium, homogenized and lyophilized. 2. The method according to claim 1, characterized in that the lyophilization is carried out in a protective medium which oine composition of 2 to 17% sodium glutamate, 5 to 20% sucrose and 0.5 to 5% of destructed gelatin.