RU2764118C1 - Polyvalent vaccine against salmonellosis of productive animals - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology. A method for manufacturing an inactivated aluminum hydroxide vaccine against pig salmonellosis is proposed, including separate cultivation in a liquid nutrient medium in fermenters of salmonella cultures of vaccine strains Salmonell choleraesuis No. 370, Salmonella typhimurium No. 371, Salmonella abortusovis No. 372 and Salmonella dublin No. 373, followed by inactivation with formaldehyde, adsorption on aluminum hydroxide, decanting clear supernatant with the preparation of a given concentration of microbial bodies in the sediment of each salmonella strain 15.0-25.0×10⁹ mb/cm³; then cultures of salmonella strains in the vaccine are mixed in equal volumes.

EFFECT: invention provides protection of productive animals from diseases of salmonella etiology.

1 cl, 1 tbl, 5 ex

Description

The invention relates to biotechnology and veterinary medicine and can be used in the industrial production of an inactivated aluminum hydroxide vaccine against salmonellosis in productive animals.

A known method of manufacturing a vaccine against paratyphoid piglets [1] and a method of manufacturing a vaccine against salmonellosis of farm animals [4].

The proposed vaccines against salmonellosis have a number of disadvantages: the cultivation process is quite long (12-14 hours), it does not allow the use of modern equipment, the protective environment for freeze-drying does not ensure the stability of the vaccine during long-term storage.

A drug based on Salmonella lysatantigens against salmonellosis in pigs is known [2, 3].

The manufacture of this drug has a number of disadvantages: Salmonella are grown in an uncontrolled mode, inactivation is carried out with hydroxylamine, which is more toxic than formalin. The drug is produced in dry form by freeze-drying. We offer a form of vaccine, as an adjuvant - aluminum hydroxide. This technology significantly reduces the cost of the vaccine.

The objective of the claimed invention is to obtain a highly immunogenic inactivated aluminum hydroxide vaccine that provides reliable protection against gastrointestinal diseases of productive animals caused by Salmonella, as well as improving the quality and standardization of the biological properties of the inactivated aluminum hydroxide vaccine.

This task is achieved by introducing aluminum hydroxide into the composition of the vaccine, as well as by the implementation of a controlled periodic cultivation process and gentle modes of Salmonella inactivation.

This vaccine contains 4 industrial strains of Salmonella - Salmonella choleraesuis 370, Salmonella typhimurium 371, Salmonella abortusovis 372 and Salmonella dublin 373 [6].

The cultivation of Salmonella is carried out as follows.

For the cultivation of Salmonella, a liquid nutrient medium based on Hottinger's digest with growth additives is used [6].

The nutrient medium in the fermenters is inoculated separately with Salmonella matted seedlings grown in a nutrient medium based on Hottinger's digest. Cultivated at (37±1)°C for 6-8 hours. After inoculation of the fermenter, the redox potential of the culture liquid is reduced to (-100) - (-120) mV by holding the culture without air supply for aeration and the mixer turned off, and then until the end of the cultivation process by changing the air flow rate for aeration and the rotation speed of the mixer the partial pressure of oxygen dissolved in the culture liquid is maintained at the level of 20-30% of the oxygen saturation of the air, the pH of the culture liquid is maintained at the level of 7.6-7.7 units. by supplying a 10% NaOH solution, and fractional supply of glucose is carried out in doses up to a concentration of (0.1-0.15)% while limiting the growth of Salmonella with glucose, characterized by a sharp increase in pO ₂ at constant air flow and stirrer speed and stopping the decrease in pH culture fluid.

The resulting bacterial culture of two industrial strains of Salmonella with a measured concentration of bacteria is pumped into four separate, pre-prepared reactors (or glass cylinders) and a fractionally 37% aqueous solution of formaldehyde is added with constant stirring to its final concentration of 0.2-0.3%, adjust the pH to 7.1-7.2 units. adding 10% NaOH solution, stirred for 15-20 min and maintained at a temperature of 37-41°C for 3-7 days.

After checking for sterility and completeness of inactivation, a formaldehyde-inactivated culture of each strain containing a known amount of microbial bodies per 1 cm ³ is added with a 4% suspension of aluminum hydroxide pH (7.2 ± 0.1) units. at the rate of 130 cm ³ for each dm ^{3 of the} culture, stirred for 15-20 minutes and kept at a temperature of 18-20°C for 2-3 days, stirring 2-3 times daily, and then without stirring for 2 days. After settling the culture adsorbed on aluminum hydroxide, the clear supernatant liquid is decanted in such a way as to obtain a concentration of (15.0-25.0)×10 ⁹ MT/cm ³ in the precipitate.

After inactivation, adsorption of cultures on aluminum hydroxide and decantation of the supernatant, the vaccine components are mixed in equal volumes.

The vaccine is packaged under aseptic conditions with constant stirring under sterile conditions.

After mixing, the vaccine is packaged under aseptic conditions with constant stirring into sterile vials.

The objective of the invention is to improve the quality of the target product by increasing the resistance of the animal organism to the disease of salmonella etiology. The proposed vaccine has a high prophylactic efficacy.

The technical result of the invention is achieved in a method for manufacturing a vaccine against salmonellosis of productive animals by including in the composition of the proposed vaccine as microorganisms the bacterial mass of four strains of Salmonella, their separate cultivation is carried out to the initial concentrations of microorganisms of 15.0-25.0 billion bw/cm ³ in fermenters at the redox potential of the culture fluid -100 ÷ -120 mV, after which pO ₂ in the culture fluid is maintained at a level of 20-30% of the oxygen saturation of the air until the end of cultivation, the pH of the culture fluid is adjusted at the level of 7.6-7 ,7, and fractional supply of glucose is carried out in doses up to a concentration of 0.1-0.15% while limiting the growth of Salmonella with glucose, inactivation of Salmonella cultures is carried out with formaldehyde at a temperature of 37-41 ° C for 3-7 days, and the final concentration of formaldehyde in the culture is 0.2-0.3%. After checking for sterility and completeness of inactivation, a formaldehyde-inactivated culture of each strain containing a known amount of microbial bodies per 1 cm ³ is added with a 4% suspension of aluminum hydroxide pH (7.2 ± 0.1) units. at the rate of 130 cm ³ for each dm ^{3 of the} culture, stirred for 15-20 minutes and kept at a temperature of 18-20 ° C for 2-3 days, stirring 2-3 times daily, and then without stirring for 2 days, after settling adsorbed on aluminum hydroxide cultures carry out decantation of a transparent supernatant to obtain a predetermined concentration of microbial bodies of each strain of Salmonella in the sediment (15.0-25.0)×10 ⁹ bw/cm ³ , then the cultures of Salmonella strains in the vaccine are mixed in equal volumes .

In the patent scientific and technical literature, technical solutions are not known that contain modes of the method for manufacturing a vaccine against salmonellosis in productive animals similar to the claimed one, i.e. the proposal meets the criterion of "novelty".

All modes of the method are feasible in industrial conditions, aimed at solving a real technical problem, i.e. sentence "industrially applicable".

This goal was achieved by selection and introduction into the vaccine of non-competing antigens of production strains and their optimal quantitative ratio, the implementation of a controlled periodic cultivation process and sparing inactivation modes.

Example 1. A method for manufacturing a vaccine with minimal parameter values

For the cultivation of Salmonella, a liquid nutrient medium based on Hottinger's digest with growth additives is used.

The medium is inoculated separately with matrix cultures of Salmonella grown in a liquid nutrient medium of the same composition. Cultivated at (37±1)°C for 6-8 hours. After inoculation of the fermenters, the redox potential of the culture fluid is reduced to -120 mV by holding the culture without air supply, after which, until the end of the cultivation process, pO ₂ in the culture fluid is maintained at a level of 20% of the oxygen saturation of the air, the pH of the culture fluid is at the level of 7.6 units by supplying a 10% NaOH solution, and fractional supply of glucose is carried out in doses up to a concentration of 0.10% while limiting the growth of Salmonella by glucose, characterized by a sharp increase in pO ₂ at constant air flow and stirrer speed and stopping the decrease in the pH of the culture liquid.

The process of inactivation of cultures of Salmonella strains is carried out with a 37% aqueous solution of formaldehyde at 37°C for 3 days, and the final concentration of formaldehyde in the culture is 0.20%.

After checking for sterility and completeness of inactivation, a formaldehyde-inactivated culture of each strain containing a known amount of microbial bodies per 1 cm ³ is added with a 4% suspension of aluminum hydroxide pH (7.2 ± 0.1) units. at the rate of 130 cm ³ for each dm ^{3 of the} culture, stirred for 15-20 minutes and kept at a temperature of 18-20°C for 2-3 days, stirring 2-3 times daily, and then without stirring for 2 days. After settling the culture adsorbed on aluminum hydroxide, the clear supernatant liquid is decanted in such a way as to obtain a concentration of 15.0 billion mt/cm ³ in the sediment.

Cultures of Salmonella strains are mixed in the vaccine in equal volumes at the following initial concentrations, billion bw/cm ³ :

- bacterial mass from the strain Salmonella choleraesuis No. 370 - 15.0;

- bacterial mass from the strain of Salmonella typhimurium No. 371 - 15.0;

- bacterial mass from the strain Salmonella abortusovis No. 372 - 15.0;

- bacterial mass from the Salmonella Dublin strain No. 373 - 15.0.

Example 2. A method for manufacturing a vaccine with optimal parameter values

For the cultivation of Salmonella, a liquid nutrient medium based on Hottinger's digest with growth additives is used.

The medium is inoculated separately with matrix cultures of Salmonella grown in a liquid nutrient medium of the same composition. Cultivated at (37±1)°C for 6-8 hours. After inoculation of the fermenters, the redox potential of the culture fluid is reduced to -110 mV by holding the culture without air supply, after which, until the end of the cultivation process, pO ₂ in the culture fluid is maintained at a level of 25% of the oxygen saturation of the air, the pH of the culture fluid is at the level of 7.6 units by supplying a 10% NaOH solution, and fractional supply of glucose is carried out in doses up to a concentration of 0.10% while limiting the growth of Salmonella by glucose, characterized by a sharp increase in pO ₂ at constant air flow and stirrer speed and stopping the decrease in the pH of the culture liquid.

The process of inactivation of cultures of Salmonella strains is carried out with a 37% aqueous solution of formaldehyde at 39°C for 5 days, with the final concentration of formaldehyde in the culture being 0.25%.

After checking for sterility and completeness of inactivation, a formaldehyde-inactivated culture of each strain containing a known amount of microbial bodies per 1 cm ³ is added with a 4% suspension of aluminum hydroxide pH (7.2 ± 0.1) units. at the rate of 130 cm ³ for each dm ^{3 of the} culture, stirred for 15-20 minutes and kept at a temperature of 18-20°C for 2-3 days, stirring 2-3 times daily, and then without stirring for 2 days. After settling the culture adsorbed on aluminum hydroxide, the clear supernatant liquid is decanted in such a way as to obtain a concentration of 20.0 billion mt/cm ³ in the sediment.

Cultures of Salmonella strains are mixed in the vaccine in equal volumes at the following initial concentrations, billion bw/cm ³ :

- bacterial mass from the strain Salmonella choleraesuis No. 370 - 20.0;

- bacterial mass from the strain of Salmonella typhimurium No. 371 - 20.0;

- bacterial mass from the strain Salmonella abortusovis No. 372 - 20.0;

- bacterial mass from the strain Salmonella dublin No. 373 - 20.0.

Example 3. A method for manufacturing a vaccine with maximum parameter values

For the cultivation of Salmonella, a liquid nutrient medium based on Hottinger's digest with growth additives is used.

The medium is inoculated separately with matrix cultures of Salmonella grown in a liquid nutrient medium of the same composition. Cultivated at (37±1)°C for 6-8 hours. After inoculation of the fermenters, the redox potential of the culture fluid is reduced to -100 mV by holding the culture without air supply, after which, until the end of the cultivation process, pO ₂ in the culture fluid is maintained at a level of 30% of the oxygen saturation of the air, the pH of the culture fluid is at the level of 7.7 units by supplying a 10% NaOH solution, and fractional supply of glucose is carried out in doses up to a concentration of 0.15% while limiting the growth of Salmonella by glucose, characterized by a sharp increase in pO ₂ at constant air flow and stirrer speed and stopping the decrease in the pH of the culture liquid.

The process of inactivation of cultures of Salmonella strains is carried out with a 37% aqueous solution of formaldehyde at 41°C for 7 days, and the final concentration of formaldehyde in the culture is 0.30%.

After checking for sterility and completeness of inactivation, a formaldehyde-inactivated culture of each strain containing a known amount of microbial bodies per 1 cm ³ is added with a 4% suspension of aluminum hydroxide pH (7.2 ± 0.1) units. at the rate of 130 cm ³ for each dm ^{3 of the} culture, stirred for 15-20 minutes and kept at a temperature of 18-20°C for 2-3 days, stirring 2-3 times daily, and then without stirring for 2 days. After settling of the culture adsorbed on aluminum hydroxide, the clear supernatant liquid is decanted in such a way as to obtain a concentration of 25.0 billion mt/cm ³ in the sediment.

Cultures of Salmonella strains are mixed in the vaccine in equal volumes at the following initial concentrations, billion bw/cm ³ :

- bacterial mass from the strain Salmonella choleraesuis No. 370 - 25.0;

- bacterial mass from the strain of Salmonella typhimurium No. 371 - 25.0;

- bacterial mass from the strain Salmonella abortusovis No. 372 - 25.0;

- bacterial mass from the strain Salmonella dublin No. 373 - 25.0.

Example 4. Method for the manufacture of a vaccine with parameter values below the minimum

For the cultivation of Salmonella, a liquid nutrient medium based on Hottinger's digest with growth additives is used.

The medium is inoculated separately with matrix cultures of Salmonella grown in a liquid nutrient medium of the same composition. Cultivated at (37±1)°C for 6-8 hours. After inoculation of the fermenters, the redox potential of the culture fluid is reduced to -130 mV by keeping the culture without air supply, after which, until the end of the cultivation process, pO ₂ in the culture fluid is maintained at a level of 15% of oxygen saturation in the air, the pH of the culture fluid is at the level of 7.2 units by supplying a 10% NaOH solution, and fractional supply of glucose is carried out in doses up to a concentration of 0.05% while limiting the growth of Salmonella by glucose, characterized by a sharp increase in pO ₂ at constant air flow and stirrer speed and stopping the decrease in the pH of the culture liquid.

The process of inactivation of cultures of Salmonella strains is carried out with a 37% aqueous solution of formaldehyde at 35°C for 1 day, and the final concentration of formaldehyde in the culture is 0.15%.

After checking for sterility and completeness of inactivation, a formaldehyde-inactivated culture of each strain containing a known amount of microbial bodies per 1 cm ³ is added with a 4% suspension of aluminum hydroxide pH (7.2 ± 0.1) units. at the rate of 130 cm ³ for each dm ^{3 of the} culture, stirred for 15-20 minutes and kept at a temperature of 18-20°C for 2-3 days, stirring 2-3 times daily, and then without stirring for 2 days. After settling the culture adsorbed on aluminum hydroxide, the clear supernatant liquid is decanted in such a way as to obtain a concentration of 15.0 billion mt/cm ³ in the sediment.

Cultures of Salmonella strains are mixed in the vaccine in equal volumes at the following initial concentrations, billion bw/cm ³ :

- bacterial mass from the strain Salmonella choleraesuis No. 370 - 15.0;

- bacterial mass from the strain of Salmonella typhimurium No. 371 - 15.0;

- bacterial mass from the strain Salmonella abortusovis No. 372 - 15.0;

- bacterial mass from the strain Salmonella dublin No. 373 - 15.0.

Example 5. Method for the manufacture of a vaccine with values above the maximum

For the cultivation of Salmonella, a liquid nutrient medium based on Hottinger's digest with growth additives is used.

The medium is inoculated separately with matrix cultures of Salmonella grown in a liquid nutrient medium of the same composition. Cultivated at (37±1)°C for 6-8 hours. After inoculation of the fermenters, the redox potential of the culture fluid is reduced to -90 mV by holding the culture without air supply, after which, until the end of the cultivation process, pO ₂ in the culture fluid is maintained at a level of 30% of the oxygen saturation of the air, the pH of the culture fluid is at the level of 7.6 units by supplying a 10% NaOH solution, and fractional supply of glucose is carried out in doses up to a concentration of 0.30% while limiting the growth of Salmonella by glucose, characterized by a sharp increase in pO ₂ at constant air flow and stirrer speed and stopping the decrease in the pH of the culture liquid.

The process of inactivation of cultures of Salmonella strains is carried out with a 37% aqueous solution of formaldehyde at 43°C for 9 days, and the final concentration of formaldehyde in the culture is 0.35%.

After checking for sterility and completeness of inactivation, a formaldehyde-inactivated culture of each strain containing a known amount of microbial bodies per 1 cm ³ is added with a 4% suspension of aluminum hydroxide pH (7.2 ± 0.1) units. at the rate of 130 cm ³ for each dm ^{3 of the} culture, stirred for 15-20 minutes and kept at a temperature of 18-20°C for 2-3 days, stirring 2-3 times daily, and then without stirring for 2 days. After settling of the culture adsorbed on aluminum hydroxide, the clear supernatant liquid is decanted in such a way as to obtain a concentration of 30.0 billion mt/cm ³ in the sediment.

Cultures of Salmonella strains are mixed in the vaccine in equal volumes at the following initial concentrations, billion bw/cm ³ :

- bacterial mass from the strain Salmonella choleraesuis No. 370 - 30.0;

- bacterial mass from the strain of Salmonella typhimurium No. 371 - 30.0;

- bacterial mass from the strain Salmonella abortusovis No. 372 - 30.0;

- bacterial mass from the strain Salmonella dublin No. 373 - 30.0.

Technological parameters for the manufacture of inactivated aluminum hydroxide vaccine against salmonellosis of pigs, indicated in the examples, are shown in the table.

The vaccine made according to examples 1-3 has a high immunogenic activity and meets the requirements of TU (STO), while the vaccine prepared according to paragraph 4 does not have sufficient immunogenic activity, and the vaccine made according to example 5 has an increased reactogenicity.

Sources of information

1. "Instructions for the manufacture and control of the vaccine against paratyphoid piglets", approved by the Main Veterinary Directorate of the USSR Ministry of Agriculture on April 18, 1985

Passport - Salmonella choleraesuis No. 370 (VGNKI)

Passport - Salmonella typhimurium No. 371 (FGBNU "VGNKI")

Passport - Salmonella abortusovis No. 372 (VGNKI)

Passport - Salmonella dublin No. 373 (VGNKI)

2. Subbotin V.V., Sidorov M.A. Immunogenic properties of lysate antigens from Salmonella, Escherichia and Staphylococcus. // Veterinary, Moscow. 1993.

3. Kudryavtsev V.V. Immunogenic properties of lysate antigens from Salmonella, Escherichia and Staphylococcus. // Abstract of dis. cand. veterinary science, Moscow. 1993.

4. Patent of the Russian Federation No. 2129016 dated April 20, 1999, A61K 39/112 "Method of manufacturing a vaccine against salmonellosis in farm animals."

5. Patent of the Russian Federation No. 2191598 dated October 27, 2002 A61K 39/102 "Associated aluminum hydroxide vaccine against infectious pneumonia of pigs of bacterial etiology and method for its manufacture."

6. Pavlenko I.V. and others. Improving the production technology of a dry live vaccine against salmonellosis in calves // Bulletin of the Kazan Technological University - 2013. No. 8 - P. 226-232.

Claims (6)

A method for manufacturing an inactivated aluminum hydroxide vaccine against salmonellosis in pigs, including inoculation of vaccine strains of Salmonella, their separate cultivation in a liquid nutrient medium in fermenters, followed by inactivation of Salmonella cultures, characterized in that the vaccine contains a bacterial mass of strains of Salmonell choleraesuis No. 370, Salmonella typhimurium adsorbed on aluminum hydroxide No. 371, Salmonella abortusovis No. 372 and Salmonella dublin No. 373, mixed in equal volumes, separately cultivated to initial concentrations, billion bw/cm ³ : bacterial mass from the strain Salmonella choleraesuis No. 370 - 15.0-25.0; bacterial mass from the strain of Salmonella typhimurium No. 371 - 15.0-25.0; bacterial mass from the strain Salmonella abortusovis No. 372 - 15.0-25.0; bacterial mass from the strain Salmonella dublin No. 373 - 15.0-25.0, at the same time, immediately after inoculation, the redox potential of the culture fluid is reduced to -120 - -100 mV, after which, until the end of the cultivation process, pO ₂ in the culture fluid is maintained at a level of 20-30% of oxygen saturation in the air, the pH of the culture fluid is adjusted at the level of 7 ,6-7.7 units, and fractional supply of glucose is carried out in doses up to a concentration of 0.1-0.15% with limiting the growth of Salmonella with glucose; the resulting bacterial culture of four industrial strains of Salmonella with a measured concentration of bacteria is pumped into four previously prepared reactors or glass bottles and a fractionally 37% aqueous solution of formaldehyde is added with constant stirring to its final concentration of 0.2-0.3%, the pH is adjusted to 7 .1-7.2 units adding 10% NaOH solution, stirred for 15-20 min and maintained at a temperature of 37-41°C for 3-7 days; after checking for sterility and completeness of inactivation, a formaldehyde-inactivated culture of each strain containing a known amount of microbial bodies per 1 cm ³ is added with a 4% suspension of aluminum hydroxide pH 7.2±0.1 units. at the rate of 130 cm ³ for each dm ^{3 of the} culture, stirred for 15-20 minutes and kept at a temperature of 18-20 ° C for 2-3 days, stirring 2-3 times daily, and then without stirring for 2 days, after settling adsorbed on aluminum hydroxide cultures carry out decantation of a transparent supernatant to obtain a predetermined concentration of microbial bodies of each strain of Salmonella in the sediment 15.0-25.0×10 ⁹ bw/cm ³ , then the cultures of Salmonella strains in the vaccine are mixed in equal volumes.