CS252537B1 - Inactivated vaccine against neonatal calf diarrhea - Google Patents

Abstract

The solution concerns the technological preparation of an inactivated vaccine against neonatal coliform diarrhea in calves 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 strains of Escherichia coli CCM 3908, CCM 3909, CCM 3910 and CCM 3911, which are technologically stable under defined conditions, chemically inactivated and equalized in the same volume of 1 X 1010 to 3 X 1010 in 1 ml of vaccine. The inactivated vaccine is applicable in immunoprophylaxis of colic in calves, where it significantly reduces morbidity and mortality of calves due to this infectious disease.

Description

The solution concerns the technological preparation of an inactivated vaccine against neonatal coli diarrhea in calves 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 strains of Escherichia coli CCM 3908, CCM 3909, CCM 3910 and CCM 3911, chemically inactivated and equalized in the same volume amount of 1 X 10 ¹⁰ to 3 X 10 ¹⁰ in 1 ml of vaccine.

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

252337

The invention relates to a method for producing an inactivated vaccine against neonatal coliform diarrhea in calves, which is technologically prepared from Escherichia coli K99+ strains, in which the quantitative expression of adhesin testing on horse and sheep erythrocytes is optimized during submerged cultivation, using mannose-resistant hemagglutination, which can be used for the specific prevention of coliform diarrhea in calves.

E. coli strains isolated from calves diagnosed with “neonatal colienteritis” produce K99 ⁺ adhesion antigens, which colonize the small intestine of calves. The expression of colonizing factors is a process regulated by the genetic material of the cell. Microbial cells during growth in vivo and in vitro exhibit qualitative and quantitative phase variation — ISAACSON and RICHTER: Escherichia coli 987P pilus: Purification and partial characterization, J. Bacteriol. 146, pp. 874—889; JONES GW, ISAACSON RE: Proteinaceus bacterial adhesine and their receptors. Brit. Rev. Microbio ¹ . 10, 1983, pp. 229—260.

For the technological production of effective immunogens against the above disease, quantitative phase variation of production strains of E. coli is particularly important. This phenomenon in vitro is regulated under submerged cultivation conditions, which are different from the conditions for qualitative phase variation. Quantitative phase variation, which determines the maximum production of adhesins, is a primary condition for the technological production of effective immunogens applicable to specific prophylaxis of diarrheal diseases in calves.

The current state of knowledge in the field of bacterial genetics and etiopathogenicity of E. coli has not utilized the phenomenon of quantitative phase variation in the production of effective substances useful in immunoprophylaxis of this disease. Utilizing this phenomenon in the field in question will ensure synchronization of adherence antigen expression throughout the entire submerged production cultivation of E. coli K99+.

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

Casamino acids 1.0 g Dihydrogen phosphate potassium 1.36 g Sodium hydrogen phosphate 10.10 g Glucose 1.0 g Calcium chloride dihydrate 0.0004 About Magnesium sulfate heptahydrate 0.010 g Ferric chloride tetrahydrate 0.000135 g Magnesium chloride 0.001 g Distilled water up to 1,000 ml

The authors experimentally verified various types of production nutrient media that would ensure maximum expression of the adhesion antigen. From the point of view of the effectiveness of quantitative expression of adhesion antigens, it was experimentally proven that the new production medium developed by the authors ensures quantitatively higher production of adhesin and its primary component is acid casein hydrolysate.

The authors of the invention isolated and, based on the phenotypic expression, determined the adherence properties of many strains of E. coli, which came from calves with acute occurrence of neonatal colienteritis. By selecting the targeted properties of the isolated strains and testing these strains in submerged cultivation, four strains were selected, deposited in the Czechoslovak collection of microorganisms of the University of JEP Brno, tl'. Obránců míru 10 under the numbers CCM 3908, CCM 3909, CCM 3910 and CCM 3911. The mentioned strains, under the cultivation conditions defined by the authors, met the required parameters, which were set for the technological production of an effective vaccine.

Mannose-resistant hemagglutination was used to test the quantitative phase of culture growth in terms of the production of adhesion antigens. To characterize the qualitative phase of growth, the authors used the slide agglutination BRUSH BORDER test and growth on Simons-citrate agar with adonite.

To ensure positivity in these reactions, the authors developed a new type of production medium with the following composition:

Acid casein hydrolysate 3—15

Potassium dihydrogen phosphate 1.36

Sodium hydrogen phosphate 10.10

Glucose 0.0—1.0

Calcium chloride dihydrate 0.004

Magnesium sulfate heptahydrate 0.10

Ferric chloride hexahydrate 0.000135

Magnesium chloride tetrahydrate 0.001

Distilled water up to 1,000 ml g

Q

pg g

g g

After the completion of laboratory verification experiments, an oral, chemically inactivated E. coli K99 ⁺ vaccine was prepared, which the authors tested in practical breeding conditions. Vaccination was carried out in February, March and April 1985. A total of 294 calves were vaccinated. Statistics from December 1984 and January 1985 served as a control. The mortality of calves after the start of vaccination was reduced by

19.5%, the number of diagnosed diarrhea decreased by 50.2%, while no coliform diarrhea was diagnosed, and the decrease in mortality was 71.8%.

It follows from the above that the invention will find wide application in cattle breeding in reducing the incidence of neonatal cholinergic enteritis. The application of this preparation is simple, as the vaccine can be administered in colostrum or milk.

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Example 1

The enteropathogenic strain Escherichia coli CCM 3908 is cultivated in 300 ml Erlenmeyer flasks containing 20 ml of sterile medium, the composition of which is on page 2 and which contains 0.5% acid casein hydrolysate and is glucose-free. The subsequent incubation is carried out for 4 hours at 37 °C with intensive stirring. After this incubation, a volume of 20 ml of the culture is transferred to a 4,000 ml Erlenmeyer flask containing 300 ml of sterile medium and incubated for 3 hours at 37 °C with intensive stirring. The subculture prepared in this way is used to inoculate the fermenter in a ratio of 300 ml of subculture to 7 liters of the sterile medium mentioned above. Submerged cultivation is carried out at 37 °C with constant stirring at 600 rpm and arrest with sterile air at 8 1/min. During the 8-hour cultivation, culture samples are taken for testing of bacterial sterility, pH value, density and detection of adhesion antigen. The resulting bacterial mass does not contain any contaminating microorganisms, with a resulting density of 1.0 measured on Specola against a reference sample at 540 nm with a positive agglutination reaction with monospecific K99 ⁺ antiserum.

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

The resulting vaccine must be free of foreign bacterial and fungal microflora and is harmless to mice after oral vaccination with 0.5 ml of vaccine. A 20-fold dilution of the vaccine must show a positive agglutination reaction with monospecific K99 ⁺ antiserum.

Example 2

The enteropathogenic strain Escherichia coli CCM 3911 was cultivated in 300 ml Erlenmeyer flasks containing 20 ml of sulfuric medium, the composition of which is on page 2 and which contains 1.5% acid casein hydrolysate and 0.1% glucose. Further technological preparation and processing of the biomass of the above strain is analogous to that given in example 1.

Example 3

The enteropathogenic strain Escherichia coli CCM 391.0 was cultivated in 300 ml Erlenmeyer flasks containing 20 ml of sterile medium, the composition of which is on page 2 and which contains 0.5% acid casein hydrolysate and 0.1% glucose. Further technological preparation and processing of the biomass of the said strain is analogous to that given in example 1.

Example 4

The enteropathogenic strain Escherichia coli CCM 3909 was cultured in 300 ml Erlenmeyer flasks containing 20 ml of the medium, the composition of which is on page 2 and which contains 0.3% acid casein hydrolysate and 0.1% glucose. Further technological preparation and processing of the biomass of the 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.