CS252537B1 - Inactivated vaccine against calves' neonal diarreal colic - Google Patents

Abstract

The solution concerns technological preparation inactivated neonatal co-vaccine diarrhea of calves in the early postnatal period. In treated calves immediately after birth induces a state of specific protection with use competitive and local immunity. vaccine contains 4 enteropathogenic techno'ogically stable conditions under defined conditions strain Escherichia coli CCM 3908, CCM 3909, CCM 3910 and CCM 3911, chemically inactivated and equalized in the same volume 1 X 1010 to 3 X 10 10 in 1 ml vaccine. The inactivated vaccine is useful in immunoprophylaxis Calf calves, where significantly reduces the morbidity and mortality of calves due to this infectious disease.

Description

The present invention relates to the technological preparation of an inactivated vaccine against neonatal coli diarrhea in the early postnatal period. In treated calves, immediately after birth, it induces a state of specific protection using competitive and local immunity. The vaccine contains 4 enteropathogenic technically, under defined conditions, stably strains of Escherichia coli CCM 3908, CCM 3909, CCM 3910 and CCM 3911, chemically inactivated and leveled in equal volumes of 1 X 10 ¹⁰ to 3 X 10 ¹⁰ per ml of vaccine.

The inactivated vaccine is useful in immunoprophylaxis of the calf whisper, where it significantly reduces the morbidity and mortality of calves due to this infectious disease.

252337

The invention relates to a process for the production of an inactivated neonatal coli diarrhea vaccine which is technologically prepared from strains of Escherichia coli K99 + in which submerged culture is optimized for the quantitative expression of adhesin by testing on equine and ram erythrocytes using manageable resistant hemag utin specific prevention of calf colienteritis.

E. coli strains isolated from calves diagnosed as "neonatal colienteritis" produce adherence antigens K99 ⁺ , which carry out colonization of the small intestine of calves. Expression of colonizing factors is a process regulated by the genetic material of a cell. Microbial cells show qualitative and quantitative phase variation during in vivo and in vitro growth - ISAACSON and RICHTER: Escherichia coli 987P pilus: Purification and partial characterization, J. Bacteriol. 146, p. 874-889; JONES GW, ISAACSON RE: Proteinaceus bacterial adhesins and their receptors. Brit. Rev. Microbio ¹ . 10, 1983, p. 229-260.

Quantitative phase variation of E. coli production strains is particularly important for the technological production of effective immunogens against said disease. This in vitro phenomenon is regulated under submerged culture conditions that are different from qualitative phase variation conditions. Quantitative phase variation, which conditions maximum production of adhesins, is a prerequisite for the technological production of effective immunogens useful for specific prophylaxis! diarrhea diseases of calves.

The prior art in the field of bacterial genetics and etiopathogenicity of E. coli has not exploited the phenomenon of quantitative phase variation in the production of effective agents useful in the immunoprophylaxis of this disease. Utilizing this phenomenon in the subject area, it will ensure synchronization of adherence antigen expression during all submerged production of E. coli K99 +.

The general trend for K99 production ^! E. coli vaccines is the cultivation of production strains in a production medium whose principal component is Casamino acids (DIFCO3.

Casamino acids 1.0 g dihydrogen potassium 1.36 g Sodium hydrogen phosphate 10.10 g glucose 1.0 g Calcium chloride dihydrate 0.0004 ABOUT Magnesium sulphate heptahydrate 0,010 g Ferric chloride tetrahydrate 0.000135 g Magnesium chloride 0,001 g Distilled water up to 1000 ml

The authors experimentally verified various types of production nutrient media that would ensure maximum expression of the adherence antigen. In terms of the efficiency of quantitative expression of adherence antigens, it has been experimentally demonstrated that autorml developed new production medium provides a quantitatively higher production of adhesin and its primary component is an acid casein hydrolyzate.

The inventors have isolated and determined the adherence properties of many E. coli strains derived from calves with an acute incidence of neonatal colienteritis based on phenotypic expression. By selecting the targeted properties of the isolated strains and testing these strains in submerged culture, four strains deposited in MS were selected. collection of microorganisms University of JEP Brno, tl '. Defenders of peace 10 under the numbers CCM 3908, CCM 3909, CCM 3910 and CCM 3911. The strains mentioned under the culture conditions defined by the author met the required parameters that were determined for the technological production of an effective vaccine.

Mango-resistant hemagglutination was used to test the quantitative phase of culture growth for the production of adherence antigens. In order to characterize the qualitative growth phase, the BRUSH BORDER test agglutination and the growth on the Adonsite Simons Citrate Agar were used by the authors.

In order to ensure positivity in these reactions, the authors have developed a new type of production medium of the following composition:

Acid casein hydrolyzate 3—15

Potassium dihydrogen phosphate 1.36

Sodium hydrogen phosphate 10,10

Glucose 0.0 - 1.0

Calcium chloride dihydrate 0.004

Magnesium sulphate heptahydrate 0,10

Ferric chloride sextahydrate 0.000135

Magnesium chloride tetrahydrate 0.001

Distilled water to 1000 ml g

Q o

pg g

g g

Upon completion of laboratory validation experiments, an oral, chemically inactivated E. coli K99 ⁺ vaccine was prepared and tested by the authors under practical breeding conditions. Vaccination was carried out in the months of February, March and April 1985. A total of 294 calves were vaccinated. Statistics of December 1984 and January 1985 served as controls. The deaths of calves after vaccination were reduced by

19.5%, the number of diagnosed diarrhea decreased by 50.2%, while no diarrhea was diagnosed and the mortality drop was 71.8 feathers.

Accordingly, the invention finds wide application in cattle breeding to reduce the incidence of neonatal colieneritis. Application of this preparation is simple, since the vaccine can be administered in colostrum or in milk.

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Example 1

The enteropathogenic strain of Escherichia coli CCM 3908 is cultured in 300 ml Erlenmeyer flasks containing 20 ml of sterile medium, the composition of which is shown on p. 2 and which contains 0.5% acid casein hydrolyzate and is glucose-free. Subsequent incubation is for 4 hours. at 37 ° C with vigorous stirring. After this incubation, transfer 20 ml of culture to a 4,000 ml Erlenraayer flask containing 300 ml of sterile medium and incubate for 3 hours. at 37 ° C with vigorous stirring. The subculture thus prepared is used to inoculate a fermenter at a rate of 300 ml of subculture per 7 liters of the sterile medium mentioned above. Submerged culture is carried out at 37 ° C with stirring at 600 rpm. and sterile air arrest 8 L / min. During the 8-hour culture, culture samples are harvested to test for bacterial sterility, pH, density, and detection of the adherence antigen. The resulting bacterial mass does not contain any contaminating microorganisms, resulting in a density of 1.0 measured on the specimen against a 540 nm reference sample with a positive agglutination reaction with the monospecific K99 ⁺ antiserum.

The bacterial culture is chemically inactivated by adding 5% aqueous formaldehyde solution to a final concentration of 0.050-0.090 g per 100 ml. We level the inactivated culture so that every strain in the same volume is represented in the vaccine. The final concentration of inactivated microorganisms in the equalized bacterial culture is 1-3 X 10 ¹⁰ .

The resulting vaccine must not contain foreign bacterial and fungal microflora and is harmless to the mouse after oral vaccination with 0.5 ml of vaccine. A 20-fold dilution of the vaccine must show a positive agglutination reaction with the monospecific K99 ⁺ antiserum.

Example 2

The enteropathogenic strain of Escherichia co and CCM 3911 was cultured in 300 ml Erlenmeyer flasks containing 20 ml of sulfuric medium, the composition of which is shown on p. 2 and containing 1.5% acid casein hydrolyzate and 0.1% glucose. Further technological preparation and processing of biomass of said strain is analogous to that given in Example 1.

Example 3

The enteropathogenic strain of Escherichia coli CCM 391.0 was cultured at 300 m! Erlenmeyer flasks containing 20 ml of sterile medium on page 2 and containing 0,5% acid casein hydrolyzate and 0,1% glucose. Further technological preparation and processing of biomass of said strain is analogous to that given in Example 1.

Example 4

The enteropathogenic strain of Escherichia coli CCM 3909 was cultured in 300 ml Erlenmeyer flasks containing 20 ml of sterile medium composition on page 2 and containing 0.3% acid casein hydrolyzate and 0.1% glucose. Further technological preparation and processing of biomass of said strain is analogous to that given in Example 1.

Claims (3)

Example 1 An enteropathogenic strain of Escherichia coliCCM 3908 was cultured in 300 ml Erlenmeyer flasks containing 20 ml of sterile medium, the composition being on page 2 containing 0.5% acid casein hydrolysis and free of glucose. Subsequent incubation was carried out for 4 hours at 37 ° C with vigorous stirring. After this incubation with a volume of 20 ml, the cultures are transferred to 4,000 ml of Erlenbacher flask containing 300 ml of sterile medium and incubated for 3 hours at 37 ° C and vigorously stirred. The subculture thus prepared is used to inoculate ferments in a ratio of 300 ml of subculture to 7 liters of sterile medium of the aforementioned. and arresting with light air 8 1 / min. During the 8-hour culture, culture samples are taken for testing the bacterial sterility, pH value, density and detection of the adhering anti-gene. The resulting bacterial meat does not contain any contaminants of microorganisms, resulting in a density of 1.0 Spekole versus a reference sample of 540nm with a positive agglutination rate of specific K99 + antisera. Bacterially inactivate the culture by adding 5% aqueous formaldehyde solution to give a final concentration of 0.050 to 0.090 g per 100 ml. Inactivate the culture so that each strain in the same population is represented in the vaccine. The final concentration of inactivated microorganisms in equalized bacterial culture is 1-3 X 1010. The resulting vaccine must not contain bacterial bacterium! and fungal microflora and is harmless to the mouse following oral vaccination of 0.5 ml of vaccine. 20-fold dilutions of the vaccines must show a positive agglutination reaction with monospecific K99 + anterior. Example 2 Enteropathogenic strain of Escherichia co iCCM 3911 was cultured in 300 ml Erlen-Mayer flasks containing 20 ml sterile medium whose composition is on page 2 and which contained 1.5% acid casein hydrolysis and 0.1% glucose. The additional preparation and processing of the bio-mass of said strain is analogous to that described in Example 1. Example 3 The enteropathogenic strain of Escherichia coliCCM 391.0 was cultivated at 300 m! Erlen-Mayer flasks containing 20 ml of sterile medium, the composition of which is on page 2, and which contains 0.5% of acidic casein hydrolyzate and 0.1% of glucose. The further biochemical preparation and processing of this strain is analogous to that described in Example 1. Example 4 The enteropathogenic strain of Escherichia coliCCM 3909 was cultured in 300 ml Erlen-Mayer flasks containing 20 ml of sterile medium whose composition is on the side. 2 and containing 0.3% acidic hydrolyzate and 0.1% glucose. Another technological preparation and processing of the biomass of said strain is analogous to that given in Example 1. P R E D Μ E T An inactivated vaccine against neonatal coli diarrhea comprising 1 X 10 10 to 3 X 10 10 inactivated strains of Escherichia coli CCM 3908, CCM 3909, CCM 3910, CCM 3911 in 1 ml vaccine. 2. The inactivated vaccine of claim 1 is characterized in that the strains of the microorganism vaccine are at least 90% purified. 3. Inactivated vaccine according to items 1 and 2, characterized in that the individual strains of Escherichia coli are represented in the vaccine in the same volume ratio.