RU2265451C2 - Method for obtaining prophylactic preparations against respiratory mycoplasmosis in poultry and method for prophylaxis - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: the present innovation deals with growing mycoplasma culture upon nutritive medium containing cholesterol-bearing liposomal emulsion as growth stimulant and bovine serumal albumin as protein source at the following ratio of ingredients, weight%: cholesterol-bearing liposomal emulsion 0.3-1.2; bovine serumal albumin 0.8-1.0; peptone 0.8-1.2; (50%) yeast extract 8.0-10.0; glucose (40%) 0.5-1.0; phenol red (indicator) (1%) 0.02-0.025; penicillin 200 U/cu. cm 10000 -20000; decoction of cardiac muscle - the rest. One should grow mycoplasma culture due to applying the strain S₆ Mycoplasma gallisepticum at activity of 10⁷-10⁸ CFU/cu. cm, not less at 37-38 C for 4-5 d, not less followed by inactivation with theotropin for 24-48 h at its final concentration of 0.2-0.3%, moreover, nutritive medium contains 0.05% cholesterol, not less. Method for prophylaxis deals with introducing prophylactic preparation obtained due to the suggested technique for poultry aged 30-35 d intra-ocularly, intra-nasally or due to feeding at the dosage of 0.05-1.0 cu. cm/poultry individual. At unsuccessful farms one should introduce the preparation mentioned repeatedly before the onset of egg-laying period. The innovation enables to decrease stress impact upon poultry.

EFFECT: higher efficiency of prophylaxis.

1 ex, 5 tbl

Description

The present invention relates to the field of veterinary medicine, in particular to poultry farming, and in particular to methods for producing prophylactic agents against respiratory mycoplasmosis of birds and to methods of prevention.

In recent years, respiratory mycoplasmosis (PM) of birds has become widespread in industrial poultry, an infectious disease characterized by damage to the respiratory organs and chronic course.

The eradication of such infections in industrial poultry is still a big problem in the egg and meat production, since it causes a decrease in productivity of 10-20 eggs per laying hen and a 10% decrease in the live weight of broilers.

A known method for producing inactivated sorbed vaccine against respiratory mycoplasmosis of birds, developed by specialists of VNIVIP (1).

According to this method for producing a vaccine against respiratory mycoplasmosis of birds, Edward medium is used to grow mycoplasma cultures, consisting of decoction of cattle heart muscle enriched with horse serum, yeast extract and glucose, or a fish concentrate hydrolyzate medium also enriched with the above substances. To inactivate the mycoplasma culture, formalin is used in a final concentration of 0.3% relative to the formaldehyde content and is kept in a thermostat at a temperature of 37 ° C for 24 hours.

Inactivated microbial mass is mixed with an aluminum hydroxide gel to a final concentration of 1.5%. The mixture is left at a temperature of (10.0 + 0.5) ° C for 48 hours to adsorb mycoplasmas. The adsorption process occurs with thorough periodic mixing of the mixture.

The obtained prophylactic agent is used in farms that are dysfunctional for respiratory mycoplasmosis. A clinically healthy bird is vaccinated twice: at the age of 1-2 months, depending on the timing of the onset of the disease and 10-15 days before the start of egg laying.

The vaccine is administered subcutaneously in the upper third of the neck. In vaccinated chickens and turkey poults, immunity occurs after 7-10 days. Immunogenicity of the vaccine is at least 80%.

However, the use of Edward’s medium containing 20% blood serum with an unstable chemical composition and the ability of blood serum proteins to adsorb on the cell membrane of mycoplasma cells during the cultivation of Mycoplasma gallisepticum (M.G.) in this method leads to unstable serum lipids and cholesterol. In addition, this method of vaccination is quite time-consuming and requires large labor costs during vaccination.

In recent years, in the United States and other countries, work has been carried out to create liposome-adjuvant vaccines, which culminated in the creation of a number of commercial biological products (2).

Closest to the proposed method is a method for producing liposome-adjuvant Mycoplasma gallisepticum vaccines containing different adjuvants for vaccination of laying hens (3). The method includes the cultivation of M. gallisepticum (M.G.) organisms, followed by their mixing with multilayer positively charged (MDS) liposomes or an oil emulsion. Vaccination of chickens is carried out twice subcutaneously at 13 and 17 weeks of age.

However, this method of obtaining the Mycoplasma gallisepticum vaccine is unstable, moreover, the method of administering the obtained vaccine is rather laborious due to the need for individual administration of the vaccine in large flocks of poultry.

The problem to be solved within the framework of the claimed group of inventions is the development of a more universal and technologically simpler way to obtain a prophylactic agent against respiratory mycoplasmosis of birds while maintaining high immunogenic properties of the resulting preparation, as well as cheaper and less labor intensive and a method of preventing respiratory mycoplasmosis in large herds of birds while reducing stress on the bird.

The problem is solved by creating a method for the prevention of respiratory mycoplasmosis in birds, including the cultivation of mycoplasma cultures using strain S ₆ Mycoplasma gallisepticum in a nutrient medium in the following ratio of ingredients, wt.%:

cholesterol-containing liposomal emulsion 0.3-1.2 bovine serum albumin 0.8-1.0 peptone 0.8-1.2 yeast extract (50%) 8.0-10.0 glucose (40%) 0.5-1.0 phenol-mouth (1% solution) 0.02-0.025 penicillin 200 units / cm ³ 10000-20000 decoction of the heart muscle rest

at a temperature of 37-38 ° C for at least 4-5 days, followed by inactivation of the obtained biomass with theotropin in its final concentration of 0.2-0.3% for 24-48 hours, and the cholesterol content in the nutrient medium is at least 0.05%.

The content of cholesterol in the nutrient medium in an amount of not less than 0.05% is due to the fact that Mycoplasma gallisepticum belongs to the group of bacteria that require the composition of the nutrient medium, and therefore, cultivation is performed on media enriched with proteins, cholesterol and vitamins.

The inventive method of prevention includes the introduction of a prophylactic agent obtained according to the method, to a bird once at the age of 30-35 days intraocularly, intranasally or by drinking in a dose of 0.05-1.0 cm ³ per head, and in dysfunctional farms, the drug is administered to the bird again before starting oviposition.

The introduction of smaller quantities does not provide reliable prevention; the introduction of large quantities is ineffective for economic reasons. As a rule, the indication for the use of this prophylactic is the failure of farms for respiratory mycoplasma of birds (serological tests are carried out for the presence of antibodies to the causative agent of respiratory mycoplasmosis in blood serum of different age groups).

Compared with conventional methods, the claimed method of prevention provides a reduction in labor costs, an increase in the rate of vaccination and a decrease in the consumption of the drug, as well as a decrease in the stress state of the bird.

The use in the claimed method of obtaining funds for the prevention of respiratory mycoplasmosis of birds of a nutrient medium of the specified composition leads to a stable and active growth of mycoplasmas, which in turn has a positive effect on the immunogenic properties of the resulting drug.

The use of artificial cholesterol-containing liposomal preparations in a nutrient medium for growing mycoplasmas makes it possible to standardize and unify the method for producing a drug and give it immunomodulating properties.

The essence and practical applicability of the claimed invention is illustrated by the following examples.

Production strains of S ₆ (Mycoplasma gallisepticum) are stored in the museum of reference mycoplasma strains in VNIVIP with passports characterizing their properties. They are stored in a lyophilized state in ampoules at a temperature of 4-6 ° C. Shelf life 12 months. The activity of strain S ₆ (Mycoplasma gallisepticum) is not less than 10 ⁷ -10 ⁸ CFU / cm ³ .

To prepare the nutrient medium, a cholesterol-containing liposomal emulsion was used, which is a milky white liquid with vesicles of an average diameter of 0.1-0.5 microns. The quantitative composition of the vesicles is 22-24 mg / ml. The cholesterol content in the liposome emulsion is not less than - 0.02822 g / g.

Example 1

Three samples of culture media were prepared for growing mycoplasmas at various component contents. For control, mycoplasmas were also cultured on Edward's medium containing growth components. 10 cm ^{3 of} seed material of the reference strain S ₆ Mycoplasma gallisepticum (M.G.), which has cultural-morphological and biochemical properties appropriate for mycoplasmas, was introduced into the prepared nutrient media (samples 1, 2, 3). Flasks were incubated in a thermostat at 37.0-37.5 ° C for 4-5 days. After 4-5 days of cultivation, a color change and turbidity of the medium was observed, indicating the growth of microorganisms. The results are presented in table 1.

Table 1.
The dependence of the output of colony forming units (CFU) of mycoplasmas on the composition of the nutrient medium. No. The content of the components of the nutrient medium, wt.% Sample No. 1 Sample No. 2 Sample No. 3 The control 1. Liposomal cholesterol-containing emulsion 0.3 0.9 1,2 50% yeast extract 8.0 9.0 10.0 Bovine serum. albumin (10%) 0.8 0.9 1 Glucose (40%) 0.5 0.7 1 Peptone 0.8 1 1,2 Phenol-mouth (1%) 0.02 0,022 0,025 Penicillin 200 U / cm ³ 10,000 10,000 2 × 10000 Decoction of the heart muscle The rest is up to 100 cm ³ 2. Mycoplasma growth (log CFU / cm ³ ) 10 ⁷ 10 ⁸ 10 ⁸ 10 ⁸

From table 1 we see that the optimal amount of liposomal emulsion is 0.3-1.2%, and bovine serum albumin is 0.8-1.0%. The number of colony forming units (CFU) of Mycoplasma gallisepticum (MG) was 10 ⁷ -10 ⁸ / cm ³ in the experiment and 10 ⁸ / cm ³ in the control.

The inactivation of the pathogen was carried out with the drug "theotropin" in a final concentration of 0.2-0.3% for 24-48 hours. Then, the samples of the obtained prophylactic agent were subject to check for sterility, safety and completeness of inactivation of mycoplasmas according to GOST (1).

The prepared samples of the preparation were sterile, harmless to chickens, had immunogenic activity, mycoplasmas were inactivated.

To study the immunogenic properties used an experimental series of the obtained drug.

In the experiment, there were four groups of chickens of 25 days of age, 10 animals each.

The first group of chickens was injected with the obtained liposome preparation against respiratory mycoplasmosis of birds at a dose of 1 cm ³ subcutaneously in the middle third of the neck.

The second group of chickens was given a vaccine against respiratory mycoplasmosis of birds and inactivated sorbed (VNIVIP) at a dose of 1 cm subcutaneously in the middle third of the neck. (1) The third group of chickens is control unvaccinated, infected (M.O.).

The fourth group of chickens is control unvaccinated and not infected.

Before setting the experiment, all chickens were bled and examined in a serum-drop reaction of agglutination (SKRA) with the color mycoplasma antigen VNIVIP. The blood serum of chickens of all groups gave negative indicators.

In experimental chickens, cellular immunity was determined on the fifth, tenth and fifteenth day after vaccination. The results of the determination of cellular immunity are presented in Table 2.

Table 2.
The results of the study of cellular immunity (E = ROCK%) Group name E = ROCK% 5th day 10th day 15th day 1st experienced 33.00 28.00 20.50 2nd control 28.50 24.00 17.00 3rd control 16.00 17.00 16.00 4th control 16.00 16.00 16.00

It was established that cellular immunity is more pronounced in chickens of the first group and amounted to 33.0% on the 5th day, 28.0% on the 10th day and 20.5% on the 15th day. In chickens of the second group, the indicators were respectively - 28.5%, 24.0% and 17.0%. From Table 2 it is seen that the drug obtained according to the proposed method has a more pronounced manifestation of cellular immunity, which, respectively, on the 5th day of the study - by 4.5%, on the 10th day - by 4.0%, by 15- e day - 3.5% higher compared to the control. Humoral immunity was determined by examining the blood serum of chickens in SKRA on the 5th, 10th, 20th, 40th and 60th days after immunization. On the 60th day after immunization, the chickens of the first 2 groups were infected intravenously with M.G. at a dose of 1.0 cm ³ . The results of determining humoral immunity are presented in Table 3. As a result, it was found that the titer of antibodies in the blood serum of the first 2 groups of chickens increased up to 20 days and lasted up to 60 days. The immunized chickens of the first 2 groups on the 21st day after infection with MG remained alive, without the manifestation of clinical signs. In chickens of the 3rd group (control unvaccinated and uninfected), the clinical signs of the disease were not manifested - (SKRA was negative all days of the study).

Table 3.
The results of the study of humoral immunity (SCRA) Group name Serum-drip agglutination reaction (SCRA) 5th day 10 - day 15th day 20th day 40th day 60th day after infected. 21 - day got sick 1 - experienced 1.4 2,4 3.0 3.2 3 2 2.2 4.0 0 2 - control 1,6 2.2 3.2 3.4 3.4 2.0 4.0 0 3 - control 0 0 0 0 0 0 4.0 5 4 - control 0 0 0 0 0 0 0 0

At the end of the experiment, chickens were examined for the release of mycoplasmas. Culture M.G. isolated only from chickens of the 3rd group - (control infected M.G.).

Thus, the drug obtained according to the claimed method has immunogenic properties, the duration of immunity is 60 days (observation period).

For the prevention of respiratory mycoplasmosis, the resulting preparation is used in egg and meat farms, for which they are administered to a poultry at the age of 30-35 days once (in successful farms), or again before the laying of eggs (in poor farms) intraocularly, intranasally, or by feeding to a dose of 0.05-1.0 cm ³ per head.

The influence of the methods of administering an agent for the prevention of respiratory mycoplasmosis in birds on the manifestation of cellular and humoral immunity was studied in 6 groups of chickens 25-30 days old, formed by 15 goals in each group. The results of the study are presented in Table 4 and Table 5, respectively.

As you can see, the proposed method for the prevention of respiratory mycoplasmosis of birds is simple, economical and convenient for use on a large number of birds.

Table 4.
Studying the influence of methods of administration of an agent for the prevention of respiratory mycoplasmosis in birds on cellular immunity. Group number The method of administration of a prophylactic agent Dose (cm ³ ) E-ROCK,% 5th day 10th day 15th day 1. Intraocular 0.05 39.0 31,0 23.0 2. Intranasally 0.05 32,0 28.5 21.5 3. Soldering 1,0 36.0 31.5 21.0 4. Control (vaccine inactive. Against RM (VNIVIP) 1,0 30,0 26.0 25.0 5. Control (unvaccinated infected) 0 23.5 21.0 21.0 6. Control (unvaccinated uninfected) 0 23.0 21.0 21.0

Table 5.
Studying the influence of methods of administration of an agent for the prevention of respiratory mycoplasmosis of birds on humoral immunity. No. Route of administration Dose, cm ³ Study days (SKRA log 21) 21st day after immunization After infection got sick fell 14th day 21st day 40th day 1. Intraocular 0.05 5,0 5,0 4.9 4.8 0 0 2. Intranasally 0.05 4,5 4.4 4.6 4,5 0 0 3. Soldering 1,0 4,5 4.7 4.8 4.6 0 0 4. Control (inactivation vaccine. (VNIVIP) 1,0 3,1 3.8 3.8 3.8 0 0 5. Control (no vaccine-infected) 0 0 3.8 3.8 3.2 0 0 6. Control (not vaccinated. Uninfected) 0 0 0 0 0 0 0

The tests showed that the means for the prevention of respiratory mycoplasmosis of birds, obtained by the proposed method, has a pronounced immunogenic activity, and the method of prevention can reduce many times the cost of prevention, reduces stress on the bird.

Sources of information

1. "Inactivated sorbed vaccine." Specifications TU 9384-006-00495674., 1998

2. Muirhead. S. "Improved disease protection comes from liposome-adjuvanted vaccines." Feedstfuffs, 1988, 60.39; 12 (English) P-25238.

3. Barbour E.K., Newman J.A. "Preliminary data of effecacy of Mycoplasma gallisepticum vaccines contaning different adjuvants in laying hens" - Veterinari - Immunology and Immunopathology, 1990, Vol. 26, No. 2, p. 115-123.

Claims (5)

1. A method of obtaining a drug for the prevention of respiratory mycoplasmosis of birds, including growing a culture of mycoplasmas on a nutrient medium followed by inactivation of the obtained biomass, characterized in that the cultivation of mycoplasmas is carried out on a nutrient medium that contains a cholesterol-containing liposomal emulsion as a growth stimulator, and as a protein source - bovine serum albumin in the following ratio of components, wt.%: Cholesterol-containing liposome emulsion 0.3-1.2 Peptone 0.8-1.2 Bovine Serum Albumin 0.8-1.0 Yeast Extract (50%) 8.0-10.0 Glucose (40%) 0.5-1.0 Phenol-mouth (1%) 0.02-0.025 Penicillin in an amount providing its contents 10000-20000 units Decoction of the heart muscle Rest at a temperature of 37-38 ° C for at least 4-5 days, followed by inactivation of the resulting biomass with theotropin for 24-48 hours at a final concentration of 0.2-0.3%. 2. The method according to claim 1, characterized in that the cultivation of a mycoplasma culture is carried out using strain S ₆ Mycoplasma gallisepticum with an activity of at least 10 ⁷ -10 ⁸ CFU / cm ³ . 3. The method according to claim 1, characterized in that the nutrient medium contains at least at least 0.05% cholesterol. 4. A method for the prevention of respiratory mycoplasmosis of birds, including the introduction of a prophylactic agent, characterized in that the prophylactic agent is a preparation obtained according to any one of claims 1 to 3, which is administered to a bird at the age of 30-35 days intraocularly, intranasally or by vaping the amount of 0.05-1.0 cm ³ per head. 5. The method according to claim 4, characterized in that in dysfunctional farms the drug is administered to the bird repeatedly before the laying of eggs.