RU2246316C1 - Mixed inactivated aluminum hydroxide-containing vaccine against infectious pneumonia and salmonellosis in pigs and method for it preparing - Google Patents

Abstract

FIELD: biotechnology, vaccines.

SUBSTANCE: vaccine comprises bacterial mass of Pasteurella multocida of serovariants A, B and D, Haemophilus pleuropneumonia of serogroups 1 and 2, and streptococcus of serogroups C and R, and also lysate-anigens of salmonellae Salmonella cholerae - suis, strain № 370 and Salmonella typhimurium № 415 mixed in the definite concentration. Vaccine elicits the high immunogenicity and provides the protection of pigs against infectious pneumonia of bacterial etiology and salmonellosis.

EFFECT: valuable veterinary properties of vaccine.

3 cl, 1 tbl, 5 ex

Description

The invention relates to biotechnology and veterinary medicine and can be used in the industrial production of the associated inactivated aluminum hydroxide vaccine against infectious pneumonia and salmonellosis in pigs and in livestock for the prevention in pigs of infectious pneumonia caused by pasteurellas, hemophilic bacteria and streptococci, as well as gastrointestinal diseases caused by salmon .

A known vaccine is associated with aluminum hydroxide against infectious pneumonia of pigs of bacterial etiology and a method for its manufacture [1] is a prototype.

Also known is a preparation based on lysate antigens of salmonella against pig salmonellosis [2, 3].

The use of the associated hydroxide-aluminum vaccine against infectious pneumonia of bacterial etiology and a preparation based on lysate antigens from Salmonella strains for specific prophylaxis of pig diseases is carried out at the same time. This allows you to create a single drug for vaccination of pigs against infectious pneumonia of bacterial etiology and salmonellosis.

A known method of manufacturing a vaccine against hemophilic pleuropneumonia of pigs [4]. However, it has several drawbacks: the process of bacterial cultivation is carried out in an uncontrolled mode, and the inactivation process is carried out for a long time with an increased formaldehyde content in the culture.

Also known are methods of manufacturing vaccines against pasteurellosis [5] and animal salmonellosis [6].

The aim of the invention is the creation of a highly immunogenic associated inactivated aluminum hydroxide vaccine that provides reliable protection not only against bacterial etiology of pneumonia caused by pasteurellas, streptococci and hemophilic bacteria, but also from gastrointestinal diseases caused by salmonella, as well as improving the quality and standardization of the biological properties of the associated vaccines.

This goal is achieved by the introduction of the known vaccine [1] lysate antigens from Salmonella strains and the optimal quantitative ratio of all antigens that make up the vaccine, as well as the implementation of a controlled periodic cultivation process and gentle modes of inactivation of hemophilic bacteria.

The method is as follows.

A liquid nutrient medium based on a Hottinger digest with growth additives is loaded into a sterile fermenter.

The nutrient medium in the fermenter is seeded with a culture of hemophilic bacteria (Haemophilus (Actinobacillus) pleuropneumoniae serogroups 1 or 2) grown in a liquid nutrient medium based on Hottinger broth with growth additives and grown at 37 ± 1 ° C for 6-8 hours.

After inoculation of the fermenter, the redox potential of the culture fluid is reduced to (-80) - (- 130) mV, after which pO ₂ in the culture fluid is maintained at a level of 15-25% of atmospheric oxygen saturation until the cultivation process, the pH of the culture fluid is adjusted to the level of 7.1-7.3 units. pH, and fractional supply of glucose is carried out in doses up to a concentration of 0.1-0.2% while limiting the growth of hemophilic bacteria with glucose, which is characterized by a sharp increase in pO ₂ with constant air flow and stirrer revolutions and stopping the decrease in pH of the culture fluid.

The resulting bacterial culture of two production strains of hemophilic bacteria with a measured concentration is pumped into separate pre-prepared sterile reactors (or glass cylinders) and fractional formaldehyde (fA) is added with constant stirring to its final concentration of 0.02-0.08%, the pH is adjusted to 7.1-7.2 units pH by adding 10% sodium hydroxide solution, stirred for 15-20 minutes and kept at 36-48 ° C for 24-72 hours.

Salmonella cultivation is carried out as follows [6].

The medium is inoculated with a Salmonella culture grown in a nutrient medium based on the Hottinger pass. Cultivate at 37 ± 1 ° C for 6-8 hours. After inoculation of the fermenter, the redox potential of the culture fluid is reduced to (-110) - (- 130) mV by holding the culture without supplying air to the aeration and the agitator turned off, and then until the end of the cultivation process by changing the air flow to aeration and the speed of rotation of the agitator they maintain the partial pressure of oxygen dissolved in the culture fluid at a level of 15-25% of the oxygen saturation of the air, the pH of the culture fluid is maintained at 7.6-7.8 units. pH by supplying a 10% solution of NaOH, and fractional glucose delivery is carried out in doses up to a concentration of 0.05-0.15% while limiting the growth of salmonella glucose, characterized by a sharp increase in pO ₂ at constant air flow and stirrer speed and stopping the decrease in pH of the culture liquids.

The resulting bacterial culture of two production Salmonella strains with a measured concentration of bacteria is pumped into separate, pre-prepared reactors (or glass cylinders) and hydrochloramine hydrochloride is added at a concentration of 0.5-0.6%.

After testing for sterility and inactivation completeness of hydroxylamine hydrochloride to the inactivated culture of each strain containing a measured amount of microbial cells in 1 cm ^3, A 4% added alumina hydrate slurry was pH 7.2 ± 0.1 at the rate of 130 cm ³ per dm ³ cultures, stirred for 15-20 minutes and kept at 18-20 ° C for 2-3 days, mixing 2-3 times daily, and then without stirring for 2 days.

After sedimentation of the culture adsorbed on alumina is carried out, a transparent supernatant is decanted so as to obtain a salmonella lysate from the bacterial suspension in a sediment with a concentration of 17.5-25.5 billion mt / cm ³ .

Streptococcus and pasteurella cultures inactivated and adsorbed on alumina hydrate for vaccines are prepared as follows [1, 7].

The process of inactivation of streptococcus strains is carried out with formaldehyde at 43 ° C for 12 hours, and the final concentration of formaldehyde in the culture is 0.02%.

The process of inactivation of pasteurellum strains is carried out with formaldehyde at 45 ° C for 18-24 hours, and the final concentration of formaldehyde in the culture is 0.14%.

After checking the sterility and completeness of inactivation, a 4% suspension of alumina hydrate, pH 7.2 ± 0.1, based on 130 cm ³ for each dm ^{3 of} culture, is added to the inactivated culture of each strain containing the measured number of microbial cells in 1 cm ³ , stirred for 15-20 minutes and kept at 18-20 ° C for 2-3 days, stirring daily for 2-3 times, and then without stirring for 2 days.

After sediment adsorbed on the alumina hydrate, the culture is decanted transparent supernatant in such a way as to obtain in the sediment concentration of microbial bodies of 17.5-25.5 billion mt / cm ³ .

To the cultures of pasteurell, streptococci and hemophilic bacteria mixed in equal amounts in the vaccine at initial concentrations of 17.5-25.5 billion mt / cm ^{3 of} pasteurell and streptococci and 10.5-17.5 billion mt / cm ³ hemophilic bacteria of each strain, additionally add salmonella lysate antigens in equal volumes at the following initial concentrations, billion mt / cm ³ :

- bacterial mass from the strain Pasteurella multocida

serovariant A-No 1231 17.5-25.5;

- bacterial mass from the strain Pasteurella multocida

serovariant B-No. 681 17.5-25.5;

- bacterial mass from the strain Pasteurella multocida

serovariant D-T-80 17.5-25.5;

- bacterial mass from a strain of Haemophilus pleuropneumoniae

serogroup 1 - No. 5870 10.5-17.5;

- bacterial mass from a strain of Haemophilus pleuropneumoniae

serogroup 2 - GU-19 10.5-17.5;

- bacterial mass from a strain of Streptococcus

zooepidemicus serogroup C-P-2082 17.5-25.5;

- bacterial mass from a strain of Streptococcus suis

serogroups R - “Castle” 17.5-25.5;

- lysate antigen from strain Salmonella

choleraesuis No. 370 17.5-25.5;

- lysate antigen from strain Salmonella

typhimurium No. 415 17.5-25.5.

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

Example 1

A method of manufacturing a vaccine with minimum parameter values

For the cultivation of hemophilic bacteria, a liquid nutrient medium based on a Hottinger pass with growth additives is used.

The medium is inoculated with a culture of hemophilic bacteria grown in a liquid nutrient medium of a similar composition. Cultivate at 37 ± 1 ° C for 6-8 hours. After inoculation of the fermenter, the redox potential of the culture fluid is reduced to -130 mV by holding the culture without air supply, after which pO ₂ in the culture fluid is maintained at a level of 15% of atmospheric oxygen saturation until the cultivation process, the pH of the culture fluid is adjusted at 7, 1 unit pH by supplying a 10% solution of NaOH, and a fractional supply of glucose is carried out in doses up to a concentration of 0.1% while limiting the growth of hemophilic bacteria with glucose, which is characterized by a sharp increase in pO ₂ at constant air flow and revolutions of the mixer and stopping the decrease in pH of the culture fluid.

The process of inactivation of hemophilic bacteria strains is carried out with formaldehyde at 36 ° C for 24 hours, and the final concentration of formaldehyde in the culture is 0.02%.

The cultures of pasteurell, streptococci and hemophilic bacteria mixed in equal volumes in the vaccine at initial concentrations of 17.5 billion mt / cm ^{3 of} pasteurell and streptococcus and 10.5 billion mt / cm ^{3 of} hemophilic bacteria of each strain, in addition add salmonella lysate antigens in equal volumes at the following initial concentrations, billion mt / cm ³ :

- bacterial mass from the strain Pasteurella multocida

serovariant A-No 1231 17.5;

- bacterial mass from the strain Pasteurella multocida

serovariant B-No 681 17.5;

- bacterial mass from the strain Pasteurella multocida

serovariant D-T-80 17.5;

- bacterial mass from a strain of Haemophilus pleuropneumoniae

serogroup 1 -№5870 10.5;

- bacterial mass from a strain of Haemophilus pleuropneumoniae

serogroup 2 - GU-19 10.5;

- bacterial mass from a strain of Streptococcus

zooepidemicus serogroup C - P -2082 17.5;

- bacterial mass from a strain of Streptococcus suis

serogroups R - “Castle” 17.5;

- lysate antigen from strain Salmonella cholerae-suis No. 370 17.5;

- lysate antigen from strain Salmonella

typhimurium No. 415 17.5.

Example 2

A method of manufacturing a vaccine with optimal values of the parameters

For the cultivation of hemophilic bacteria, a liquid nutrient medium based on a Hottinger pass with growth additives is used.

The medium is inoculated with a culture of hemophilic bacteria grown in a liquid nutrient medium of a similar composition. Cultivate at 37 ± 1 ° C for 6-8 hours. After inoculation of the fermenter, the redox potential of the culture fluid is reduced to -105 mV by holding the culture without air supply, and then until the end of the cultivation process, the pO ₂ in the culture fluid is maintained at 20% of atmospheric oxygen saturation, the pH of the culture fluid is adjusted at 7, 2 units pH by supplying a 10% solution of NaOH, and a fractional supply of glucose is carried out in doses up to a concentration of 0.15% while limiting the growth of hemophilic bacteria with glucose, which is characterized by a sharp increase in pO ₂ with constant air flow and revolutions of the mixer and stopping the decrease in pH of the culture fluid.

The process of inactivation of hemophilic bacteria strains is carried out with formaldehyde at 42 ° C for 48 hours, and the final concentration of formaldehyde in the culture is 0.05%.

The cultures of pasteurell, streptococci and hemophilic bacteria mixed in equal volumes in the vaccine at initial concentrations of 21.0 billion metric tons / cm ^{3 of} pasteurell and streptococci and 14.0 billion metric tons / cm ^{3 of} hemophilic bacteria of each strain, in addition add salmonella lysate antigens in equal volumes at the following initial concentrations, billion mt / cm ³ :

- bacterial mass from the strain Pasteurella multocida

serovariant A-No 1231 21.0;

- bacterial mass from the strain Pasteurella multocida

serovariant B-No. 681 21.0;

- bacterial mass from the strain Pasteurella multocida

serovariant D-T-80 21.0;

- bacterial mass from a strain of Haemophilus

pleuropneumoniae serogroup 1 - No. 5870 14.0;

- bacterial mass from a strain of Haemophilus pleuropneumoniae

serogroup 2 - GU-19 14.0;

- bacterial mass from a strain of Streptococcus

zooepidemicus serogroup C - P -2082 21.0;

- bacterial mass from a strain of Streptococcus suis

serogroups R - “Castle” 21.0;

- lysate antigen from strain Salmonella

cholerae-suis No. 370 21.0;

- lysate antigen from strain Salmonella

typhimurium No. 415 21.0.

Example 3

A method of manufacturing a vaccine with maximum values of the parameters

For the cultivation of hemophilic bacteria, a liquid nutrient medium based on a Hottinger pass with growth additives is used.

The medium is inoculated with a culture of hemophilic bacteria grown in a liquid nutrient medium of a similar composition. Cultivate at 37 ± 1 ° C for 6-8 hours. After inoculation of the fermenter, the redox potential of the culture fluid is reduced to -80 mV by holding the culture without air supply, after which pO ₂ in the culture fluid is maintained at a level of 25% of atmospheric oxygen saturation until the cultivation process, the pH of the culture fluid is adjusted at 7, 3 units pH by supplying a 10% solution of NaOH, and a fractional supply of glucose is carried out in doses up to a concentration of 0.2% while limiting the growth of hemophilic bacteria with glucose, which is characterized by a sharp increase in pO ₂ with constant air flow and stirrer speed and stopping the decrease in pH of the culture fluid.

The process of inactivation of hemophilic bacteria strains is carried out with formaldehyde at 48 ° C for 72 hours, and the final concentration of formaldehyde in the culture is 0.08%.

The cultures of pasteurell, streptococci and hemophilic bacteria mixed in equal amounts in the vaccine at initial concentrations of 25.5 billion mt / cm ^{3 of} pasteurell and streptococci and 17.5 billion mt / cm ^{3 of} hemophilic bacteria of each strain, in addition add salmonella lysate antigens in equal volumes at the following initial concentrations, billion mt / cm ³ :

- bacterial mass from the strain Pasteurella multocida

serovariant A-No 1231 25.5;

- bacterial mass from the strain Pasteurella multocida

serovariant B-No. 681 25.5;

- bacterial mass from the strain Pasteurella multocida

serovariant D-T-80 25.5;

- bacterial mass from a strain of Haemophilus pleuropneumoniae

serogroup 1 -№ 5870 17.5;

- bacterial mass from a strain of Haemophilus pleuropneumoniae

serogroup 2 - GU-19 17.5;

- bacterial mass from a strain of Streptococcus

zooepidemicus serogroup SP-2082 25.5

- bacterial mass from a strain of Streptococcus suis

serogroups R - “Castle” 25.5;

lysate antigen from Salmonella strain

cholerae-suis No. 370 25.5;

- lysate antigen from strain

Salmonella typhimurium No. 415 25.5.

Example 4

A method of manufacturing a vaccine with parameter values below the minimum

For the cultivation of hemophilic bacteria, a liquid nutrient medium based on a Hottinger pass with growth additives is used.

The medium is inoculated with a culture of hemophilic bacteria grown in a liquid nutrient medium of a similar composition. Cultivate at 37 ± 1 ° C for 6-8 hours. After inoculation of the fermenter, the redox potential of the culture fluid is reduced to -140 mV by holding the culture without air supply, after which pO ₂ in the culture fluid is maintained at a level of 10% of atmospheric oxygen saturation until the cultivation process, the pH of the culture fluid is adjusted at 7, 0 units pH by supplying a 10% solution of NaOH, and a fractional supply of glucose is carried out in doses up to a concentration of 0.05% while limiting the growth of hemophilic bacteria with glucose, which is characterized by a sharp increase in pO ₂ with constant air flow and stirrer speed and stopping the decrease in pH of the culture fluid.

The process of inactivation of hemophilic bacteria strains is carried out with formaldehyde at 30 ° C for 12 hours, and the final concentration of formaldehyde in the culture is 0.01%.

The cultures of pasteurell, streptococci and hemophilic bacteria mixed in equal amounts in the vaccine at initial concentrations of 14.0 billion mt / cm ^{3 of} pasteurell and streptococci and 7.0 billion mt / cm ^{3 of} hemophilic bacteria of each strain, in addition add salmonella lysate antigens in equal volumes at the following initial concentrations, billion mt / cm ³ :

- bacterial mass from the strain Pasteurella multocida

serovariant A-No 1231 14.0;

- bacterial mass from the strain Pasteurella multocida

serovariant B-No. 681 14.0;

- bacterial mass from the strain Pasteurella multocida

serovariant D-T-80 14.0;

- bacterial mass from a strain of Haemophilus pleuropneumoniae

serogroup 1 - No. 5870 7.0;

- bacterial mass from a strain of Haemophilus pleuropneumoniae

serogroup 2 - GU-19 7.0;

- bacterial mass from a strain of Streptococcus

zooepidemicus serogroup C-P-2082 14.0;

- bacterial mass from a strain of Streptococcus suis

serogroup K- “Castle” 14.0;

- lysate antigen from a strain of Salmonella cholerae-

suis No. 370 15.0;

- lysate antigen from strain Salmonella

typhimurium No. 415 15.0.

Example 5

A method of manufacturing a vaccine with parameter values above the maximum

For the cultivation of hemophilic bacteria, a liquid nutrient medium based on a Hottinger pass with growth additives is used.

The medium is inoculated with a culture of hemophilic bacteria grown in a liquid nutrient medium of a similar composition. Cultivate at 37 ± 1 ° C for 6-8 hours. After inoculation of the fermenter, the redox potential of the culture fluid is reduced to -70 mV by holding the culture without air supply, after which pO ₂ in the culture fluid is maintained at a level of 30% of atmospheric oxygen saturation until the cultivation process, the pH of the culture fluid is adjusted at 7, 4 units pH by supplying a 10% solution of NaOH, and a fractional supply of glucose is carried out in doses up to a concentration of 0.25% while limiting the growth of hemophilic bacteria with glucose, characterized by a sharp increase in pO ₂ with constant air flow and stirrer speed and stopping the decrease in pH of the culture fluid.

The process of inactivation of hemophilic bacteria strains is carried out with formaldehyde at 54 ° C for 84 hours, and the final concentration of formaldehyde in the culture is 0.09%.

The cultures of pasteurell, streptococci and hemophilic bacteria mixed in equal volumes in the vaccine at initial concentrations of 28.0 billion metric tons / cm ^{3 of} pasteurell and streptococci and 21.5 billion metric tons / cm ^{3 of} hemophilic bacteria of each strain, in addition add salmonella lysate antigens in equal volumes at the following initial concentrations, billion mt / cm ³ :

- bacterial mass from the strain Pasteurella multocida

serovariant A-No 1231 28.0;

- bacterial mass from the strain Pasteurella multocida

serovariant B-No 681 28.0;

- bacterial mass from the strain Pasteurella multocida

serovariant D-T-80 28.0;

- bacterial mass from a strain of Haemophilus pleuropneumoniae

serogroup 1 - No. 5870 21.5;

- bacterial mass from a strain of Haemophilus pleuropneumoniae

serogroup 2 - GU-19 21.5;

- bacterial mass from a strain of Streptococcus

zooepidemicus serogroup C-P-2082 28.0;

- bacterial mass from a strain of Streptococcus suis

serogroups R - “Castle” 28.0;

- lysate antigen from strain Salmonella

cholerae-suis No. 370 28.0;

- lysate antigen from strain Salmonella

fyphimurium No. 415 28.0.

The technological parameters of the manufacture of the associated inactivated aluminum hydroxide vaccine against infectious pneumonia and salmonellosis of pigs, indicated in the examples, are given in the table.

The vaccine prepared according to examples 1-3 has a high immunogenic activity and meets the requirements of technical conditions, while the vaccine prepared according to example 4 does not have sufficient immunogenic activity, and the vaccine prepared according to example 5 has increased reactogenicity.

Sources of information

1. RF patent No. 2191598 of October 27, 2002, A 61 K 39/102 “Aluminum hydroxide associated vaccine against infectious pneumonia of pigs of bacterial etiology and a method for its manufacture”

2. Kudryavtsev VV Immunogenic properties of lysate antigens from salmonella, Escherichia and staphylococci. // Abstract of thesis. Cand. Vetnauk, Moscow. 1993.

3. Subbotin V.V., Sidorov M.A. Immunogenic properties of lysate antigens from salmonella, eschirichia and staphylococcus. // Veterinary medicine, 1996. No. 4.

4. Instructions for the manufacture and control of a vaccine against hemophilic pleuropneumonia of pigs emulsified, approved. 07/11/90.

5. RF patent No. 1566533 dated April 7, 1993, A 61 K 39/102 “Method for the manufacture of a vaccine against bird pasteurellosis”.

6. RF patent No. 2129016 dated April 20, 1999, A 61 K 39/112 “Method for the manufacture of a vaccine against salmonellosis of farm animals”.

7. Copyright certificate of the Russian Federation No. 1792708 dated October 8, 1992, A 61 K 39/102 “Method for the production of sera for the treatment and prevention of pasteurellosis of farm animals, mainly pigs, fur animals and rabbits”.

Claims (3)

1. Inactivated aluminum hydroxide associated vaccine against infectious pneumonia and salmonellosis of pigs, containing the bacterial mass of Pasteurella multocida serovariants A, B and D, Haemophilus pleuropneumoniae serogroups 1 and 2 and streptococci serogroups C and R, mixed in the vaccine in equal volumes of 17 at the initial concentration of 5.5 initial concentrations -25.5 billion mt / cm ^{3 of} pasteurellas and streptococci and 10.5-17.5 billion mcl / cm ^{3 of} hemophilic bacteria of each strain, characterized in that it additionally contains Salmonella salmonella cholerae lysate antigens suis strain No. 370 and Salmonella typhimurium strain No. 415, c eshannyh in equal volumes with the following initial concentrations billion bw / cm ^3: The bacterial mass of the strain Pasteurella multocida serovariant A - No. 1231 17.5-25.5 The bacterial mass of the strain Pasteurella multocida serovariant B - No. 681 17.5-25.5 The bacterial mass of the strain Pasteurella multocida serovariant D-T-80 17.5-25.5 Bacterial mass from the strain Haemophilus pleuropneumoniae serogroup 1 - No. 5870 10.5-17.5 Bacterial mass from the strain Haemophilus pleuropneumoniae serogroup 2 - GU-19 10.5-17.5 Bacterial mass from a strain of Streptococcus zooepidemicus serogroup С-П-2082 17.5-25.5 Bacterial mass from a strain of Streptococcus suis serogroup R - “Castle” 17.5-25.5 Salmonella Lysate Antigen cholerae-suis No. 370 17.5-25.5 Salmonella Lysate Antigen typhimurium No. 415 17.5-25.5 2. A method of manufacturing a vaccine associated inactivated aluminum hydroxide against infectious pneumonia and salmonellosis of pigs, including inoculation of vaccine strains of pasteurella, hemophilic bacteria, streptococci and salmonella, their separate cultivation in a liquid nutrient medium in fermenters, followed by inactivation of pasteurella cultures, hemophilic bacteria and streptococci lyside formation salmonella hydroxylamine hydrochloride, adsorption of all antigens on an adjuvant and mixing inactivated ltur, characterized in that by culturing strains of bacteria cultures hemophilic immediately after seeding the redox potential of the culture fluid is lowered to (-80) - (- 130) mV, then until the end of the cultivation process is maintained pO ₂ in the culture medium at 15 25% of the oxygen saturation of the air, the pH of the culture fluid is adjusted to 7.1-7.3, and the fractional supply of glucose is carried out in doses up to a concentration of 0.1-0.2% while limiting the growth of hemophilic bacteria with glucose. 3. A method of manufacturing a vaccine according to claims 1 and 2, characterized in that the inactivation of hemophilic bacteria cultures is carried out with formaldehyde at a temperature of 36-48 ° C for 24-72 hours, and the final concentration of formaldehyde in the culture is 0.02-0.08 %