RU2352355C1 - Combined vaccine for rabbit visceral mycoses - Google Patents

Abstract

FIELD: veterinary science.

SUBSTANCE: invention refers to veterinary microbiology. The vaccine contains soluble antigen of Mucor racemosus № 195 fungus strain as endotoxin of its cell wall and cytoplasm. Additionally the vaccine contains soluble antigen of Candida albicans № 253 fungus strain as its cell wall and cytoplasm at cultivation term 28-36 hours and fungus cell concentration 3·10⁸-4·10⁸ per 1 cm³ of distilled water. Herewith the vaccine contains endotoxin of cell wall and cytoplasm of Mucor racemosus № 195 fungus at cultivation term 48-56 hours and fungus cell concentration 7·10⁶-13·10⁶ per 1 cm³ of distilled water, in ratio as follows (wt %): antigen of Mucor racemosus № 195 fungus strain as endotoxin of its cell wall and cytoplasm at fungus cell concentration 7·10⁶-13·10⁶ per 1 cm³ of distilled water - 55-65, antigen of Candida albicans № 253 fungus strain as cell wall and cytoplasm at fungus cell concentration 3·10⁸-4·10⁸ per 1 cm³ of distilled water 35-45.

EFFECT: vaccine is effective both for candidosis and mucormycosis, being immunologically relevant and non-allergenic.

2 tbl, 1 ex

Description

The invention relates to veterinary microbiology and can be used to obtain vaccines against visceral mycoses of rabbits, in particular against candidiasis and mucorosis.

Candidiasis (candidiasis, thrush) is an infectious disease of farm animals, mainly young animals of 2 months of age, caused by pathogenic fungi from the genus Candida, characterized by damage to the mucous membranes (mainly the digestive tract) in the form of loose or compacted deposits on them. Lesions are localized in calves and lambs in the oral cavity, esophagus, pancreas, in piglets - in the stomach, intestines and less often in other organs. Birds of all types and humans also get sick.

Mucorosis (mucoromycosis, ficomycosis) is an infectious disease of farm animals, characterized by the development of a granulomatous process in the mesenteric and submandibular lymph nodes, as well as in the liver, kidneys, lungs. In young animals, the disease proceeds with the formation of ulcers, heart attacks and infiltrates in the mucous membranes of the stomach and intestines. A person is also sick.

A known vaccine against dermatomycosis (dermatophytosis) (see AS USSR No. 1734762, publ. 05.23.92) containing inactivated microconidia of virulent fungi T.mentagrophytes, M.canis and M.mentagrophytes in a ratio of 600 million - 1 billion in 1 cm ³ (1: 1: 1) - 25-26%, 0.5-0.6% formalin solution - 25-26%, 0.8-0.9% sodium nucleinate solution - 48-50%. The vaccine is administered intramuscularly twice with an interval of 10-14 days at a dose of 0.9-1.2 cm ³ .

However, this vaccine is intended for the prevention of only dermatomycosis (trichophytosis and microsporia). In addition, the vaccine has an immunomodulator in its composition - sodium nucleinate, the effect of which on immunocompetent cells can be negative.

Known vaccine Polivak-TM for the prevention and treatment of dermatophytosis in animals (see RF patent No. 2020959, publ. 10/15/94,) containing 8 different strains of Trichophytosis and microsporia pathogens, with a concentration of microconidia of 40-120 million / cm ³ and inactivated by thiomersal .

However, the vaccine creates immunity only to the causative agents of dermatomycosis. In addition, the thiomersal inactivator has toxic properties for animals.

Closest to the claimed one is a vaccine against visceral mycoses of farm animals (see RF patent No. 2270694 according to IPC class A61K 39/00, published on 02.27.2006) containing the antigen in the form of a complex of the cytoplasmic fraction and endotoxin of the cell wall of the fungus strain Mucor racemosus No. 195 with spore activity of 5 · 10 ⁵ -6 · 10 ⁵ in 1 cm ³ .

This vaccine is intended only for the formation of anti-frost immunity and cannot be used to prevent other visceral mycoses.

The invention is aimed at solving the problem of creating an effective vaccine with a universal set of lipids, polypeptides and carbohydrates against visceral mycoses, which has highly immunogenic properties and acts simultaneously against several fungal diseases.

To solve the problem, a vaccine against visceral rabies mycoses containing soluble antigen from the strain of the fungus Mucor racemosus No. 195 in the form of endotoxin of its cell wall and cytoplasm, according to the invention, further comprises a soluble antigen from the strain of the fungus Candida albicans No. 253 in the form of its cell wall and cytoplasm by cultivation for a period of 28-36 hours and the concentration of fungus cells 3 × 10 ⁸ -4 10 ⁸ 1 cm of distilled water, the vaccine contains cell wall and endotoxin cytoplasm fungus Mucor racemosus №195 cultivation period with 48-56 ca s and concentration of fungus cells 7 × 10 ⁶ -13 x 10 ⁶ in 1 cm ³ of distilled water in the following ratio (wt.%):

Mushroom strain antigen Mucor racemosus No. 195 in the form endotoxin of its cell wall and cytoplasm with cell concentration mushroom 7 · 10 ⁶ -13 · 10 ⁶ in 1 cm ³ distilled water 55-65 Mushroom strain antigen Candida albicans No. 253 in the form cell wall and cytoplasm with cell concentration mushroom 3 · 10 ⁸ -4 · 10 ⁸ in 1 cm ³ distilled water 35-45

The sources of patent and scientific and technical information known to the authors do not describe a highly effective associated vaccine against visceral mycoses, which has the necessary and sufficient set of lipids, polypeptides and carbohydrates, which would be highly immunogenic at the same time in relation to rabbit candidiasis and mucorosis.

The authors experimentally selected two types of antigens of strains of fungi C. albicans and M.racemosus, differing in the content of lipids, polypeptides and carbohydrates.

The selection of components was based on the principle of the inseparable mutually beneficial effect of two microorganisms on each other.

In particular, a small amount of lipids in the C. albicans antigen is compensated by the amount of lipids of the M.racemosus antigen.

The value of disaccharides also prevails in the antigen from the strain of M.racemosus (almost 100 times). A sufficient amount of polysaccharides was detected in C. albicans, in contrast to the antigen M.racemosus, in which they were not even found.

The number of polypeptides with a molecular weight of up to 10 kDa and the total nitrogen content everywhere prevail 7–9 times for the M.racemosus antigen. And only the number of polypeptides with a molecular weight of more than 10 kD is greater for the C. albicans antigen (almost 7 times as well).

Characterization of antigens by lipid composition, carbohydrate composition and total nitrogen content, as well as the polypeptide composition are presented in table 1.

Table 1.
Characterization of fungal antigens by lipid composition, carbohydrate composition, and polypeptide composition No. p.p. Name and composition of components Antigens M.racemosus No. 195 C.albicans No. 253 The lipid content,% one. The total lipid content,% 2.05 0.22 The carbohydrate content, mg% 2. Monosaccharides 49.1 49.5 3. Disaccharides 1379 14.0 four. Polysaccharides but 76.0 The polypeptide composition, mg% 5. Total nitrogen 28.8 3,7 6. Polypeptides with M.M.> 10 kD 12,4 86.4 7. Polypeptides with M.M. 5.0-10 kD 7.1 1,1 8. Polypeptides with M.M. 1.5-5.0 kD 21,4 2.5 9. Polypeptides with M.m. <1.5 kD 128.5 17.1

Known separately: a vaccine against candidiasis (see RF patent No. 2217163 according to class IPC A61K 39/00, published on November 27, 2003) and a vaccine against mucorosis, which is a prototype of this invention. However, these vaccines contain antigens obtained after 6 days of cultivation (for C. albicans) and after 12 days (for M. racemosus). The concentration of fungal cells (spore activity) in these antigens is different: 4.5 · 10 ⁸ -5.5 · 10 ⁸ in 1 cm ³ for C. albicans and 5 · 10 ⁶ -6 · 10 ⁶ in 1 cm ³ for M. racemosus. In this case, the cultivation of the strain of C. albicans fungus was carried out on solid nutrient medium (agar using Hank's saline solution according to RF patent No. 2111245), and M.racemosus fungus on liquid nutrient medium (Hanks broth).

In the claimed vaccine, cultivation of fungal cells (C. albicans and M.racemosus) was carried out on solid nutrient media (Saburo agar). Significant is the concentration of spores obtained at an early stage of cultivation, in which the vaccine will have immunogenicity.

For C. albicans fungus antigen, this is 3 · 10 ⁸ -4 · 10 ⁸ in 1 cm of distilled water after 28-36 hours of cultivation, and for M.racemosus it is 7 · 10 ⁶ -3 · 10 ⁶ in 1 cm ^{3 of} distilled water after 48-56 hours.

The experimentally selected ratio of the components - antigens, namely, only with the ratio of antigens: 35% -45% of the strain of the fungus C. albicans and 55% -65% of the antigen of the strain of the fungus M.racemosus, the effectiveness of the vaccine is achieved.

The foregoing allows us to conclude that there is a criterion of "inventive step" in the claimed invention.

Strain Candida albicans No. 253, intended for the manufacture of a vaccine, is characterized by the following properties.

Morphological signs. Oval (2: 1) or almost round cells with clearly visible vacuoles with small nuclei.

Cultural signs. On Saburo agar, after 48 hours of cultivation at a temperature of 28 ° C, convex white colonies are formed with a slight creamy tint of creamy consistency, with a smooth, shiny surface and even edges.

Enzymatic activity is manifested in a semi-liquid Giss medium: with glucose - the formation of acid and gas; with maltose - also acid and gas; with sucrose - only acids; activity with lactose is not manifested.

The activity of the strain is the beginning of spore formation on the 2nd day of cultivation.

Antigens of a strain - a complex of specific proteins, polysaccharides and lipids.

Strain M.racemosus No. 195, intended for the manufacture of a vaccine, is characterized by the following properties.

Cultural and morphological characters. The growth of the fungus after incubation at 37 ° C on Saburo plate agar appears after 24 hours in the form of a whitish fluffy coating. After 72 hours, a voluminous fluffy colony is formed with a powerful, well-developed grayish mycelium. By 3 days of cultivation in test tubes in Saburo broth, surface growth of the culture is observed in the form of dense “felt” disks with developed mycelium and with the simultaneous formation of a cotton-like precipitate.

During microscopy (with fixation in 10% KOH or in 50% glycerol), the fungus is represented by unseptic mycelium, which has a different width (up to 20 μm) along the thread, branching tree-like. It is detected with an increase in the microscope (x7). Sporangiophores are rectilinear, sporangia are spherical, without apophyses.

The product synthesized by the strain is mainly a complex of proteins, polysaccharides, peptidoglycans with antigenic properties

The strain belongs to the III group of pathogenicity - conditionally pathogenic.

The strain Mucor racemosus No. 195 is supported by the method of periodic reseeding on artificial nutrient media on the basis of spore formation and cultural-morphological properties. Mutagens were not used in the work with the strain.

No animal passage required.

The strain Mucor racemosus No. 195 is stored on Saburo agar for no more than 20-30 days, followed by reseeding first on Saburo broth and then on Saburo agar. The optimal method for storing the strain is in ampoules in a lyophilized state at a temperature of 4 ° C for up to 1 year.

To obtain antigens of the strain Candida albicans No. 253, which are the cytoplasmic fraction of the fungus, and antigens of the strain Mucor racemosus No. 195, which are cell membranes (endotoxin and the cytoplasmic fraction of the fungus), use the method of destruction of cell membranes of microorganisms using ultrasonic disintegration using U.F. apparatus .BUT. - 11 (Poland), “Solana” (Erian industries) or a small-sized laboratory disintegrator (ML - 1) manufactured by the Biopribor Scientific-Production Association of the USSR Academy of Sciences.

Exemption from destroyed and non-destroyed cells is carried out by the method of filtration through the membrane "Vladipor" MFA - MA No. 3.

The selection of the immunogenic stage of antigens is carried out using the leukocyte agglomeration reaction (RAL) according to the method described in RF patent No. 2176392, according to which antigens are mixed with complement of guinea pig, stabilized rabbit blood is added, the mixture is incubated, smears are prepared, a fixed number of leukocytes is calculated and note the number of agglomerated white blood cells. The magnitude of the agglomeration index assesses the suitability of the obtained antigens for creating a vaccine. Experimentally, the leukocyte agglomeration index value was selected at which the antigen is considered immunogenic. For the antigen of Candida albicans strain No. 253 in the form of a cytoplasmic fraction, the agglomeration index is 6-10%, and for antigens of the strain Mucor racemosus No. 195 it is 6-15%.

To the soluble antigens identified by the above method are added to neutralize and increase the immunogenic properties of formalin of 0.3-0.4% concentration.

The antigen from strain Candida albicans No. 253 in the form of a cell wall and cytoplasm with a concentration of fungal cells of 3 · 10 ⁸ -4 · 10 ⁸ in 1 cm of distilled water is prepared as follows.

1. Strain Candida albicans No. 253 seeded in test tubes with Saburo agar and cultured in an incubator at 28 ° C for 3 days. The number of spores should be 6 · 10 ⁸ -9 · 10 ⁸ spores in 1 cm ^{3 of} physiological saline.

2. The grown culture is washed off and sown in bacteriological mattresses with nutrient agar, cultured for 28-36 hours at 28 ° C.

3. A culture is selected with a spore yield of 3 · 10 ⁸ -4 · 10 ⁸ in 1 cm ^{3 of} distilled water.

4. Receive the cytoplasmic fraction in the form of cell walls and the cytoplasm of the fungus by the method of destruction of cell membranes: they are exposed to an ultrasonic disintegrator for 60 minutes and the filtrate is obtained using the Vladipor membrane in the form of a soluble protein-polysaccharide complex.

5. Assess the immunogenic stage of the obtained antigen by the method of agglomeration of leukocytes. Selected antigen from strain Candida albicans No. 253, in which the index of agglomeration of leukocytes is 6-10%.

The antigen from strain Mucor racemosus No. 195 in the form of endotoxin of its cell wall and cytoplasm with a concentration of fungal cells of 7 · 10 ⁶ -13 · 10 ⁶ in 1 cm ^{3 of} distilled water is prepared as follows.

1. The strain Mucor racemosus No. 195 is seeded in tubes with Saburo agar and cultured in an incubator at 28 ° C for 3 days.

2. The grown culture is washed off and inoculated into bacteriological mattresses with nutrient agar, cultured for 48-56 hours at 28 ° C.

3. A culture is selected with a spore yield of 7 · 10 ⁶ -13 · 10 ⁶ in 1 cm ^{3 of} distilled water.

4. The cytoplasmic fraction and endotoxin of the obtained cell suspension are obtained by the method of cell membrane disruption: they are exposed to an ultrasonic disintegrator for 10 minutes and the filtrate is obtained using the Vladipor membrane in the form of a soluble protein-polysaccharide complex.

5. Assess the immunogenic stage of the obtained antigen by the method of agglomeration of leukocytes. Selected antigen from strain Mucor racemosus No. 195, in which the agglomeration index of leukocytes is 6-15%.

The vaccine is prepared as follows.

1. Take the antigen from strain Candida albicans No. 253 in the form of a cell wall and cytoplasm with a concentration of fungal cells 3 · 10 ⁸ -4 · 10 ⁸ in 1 cm ^{3 of} distilled water.

2. Take the antigen from strain Mucor racemosus No. 195 in the form of endotoxin of its cell wall and cytoplasm with a concentration of fungal cells of 7 · 10 ⁶ -13 · 10 ⁶ in 1 cm ^{3 of} distilled water.

3. Two antigens are mixed in the ratio: 35% -45% Candida albicans antigen and 55-65% Mucor racemosus antigen.

The percentage of vaccine components was selected experimentally.

4. Evaluate the immunogenicity of the obtained vaccine according to the index of agglomeration of leukocytes. The vaccine is considered immunogenic with a leukocyte agglomeration index of 6-12%.

To assess the effectiveness of the vaccine, groups of animals (rabbits) were formed with 6 animals each.

The vaccine was administered intramuscularly at a dose of 3 ml per head twice with an interval of 7-10 days. A group of control animals was injected with saline.

14 days after revaccination, rabbits were infected with cultures containing mycelium and fungal spores from Mucor racemosus No. 195 and Candida albicans No. 253 strains obtained after 3 days of cultivation on solid nutrient medium containing spores:

- 6 · 10 ⁹ 1 cm ³ distilled water for Candida albicans No. 253;

- 2 · 10 ⁶ in 1 cm ^{3 of} distilled water for Mucor racemosus No. 195.

Infection was carried out intravenously in the marginal ear vein at a dose of 0.5 ml of each culture.

Animals were monitored for 30 days. The death of animals in the control groups was 100%.

Options for using different concentrations of vaccine components and their effect on efficacy are presented in table 2.

Example 1

A vaccine is prepared with the following components:

- antigen from strain Candida albicans No. 253 (with a spore concentration of 3 · 10 ⁸ -4 · 10 ⁸ in 1 cm ^{3 of} distilled water after 28-36 hours of cultivation) in an amount of 40%,

- antigen from strain Mucor racemosus (with a spore concentration of 7 · 10 ⁶ -13 · 10 ⁶ in 1 cm ^{3 of} distilled water after 48-56 hours) in an amount of 60%.

The vaccine was administered to rabbits (6 animals) intramuscularly at a dose of 3 ml per head twice with an interval of 7 days. A group of control animals was injected with saline.

14 days after revaccination, the animals were experimentally infected by introducing fungal cultures from Mucor racemosus No. 195 and Candida albicans No. 253 strains with a spore content of 6 · 10 ⁹ in 1 cm ^{3 of} distilled water for Candida albicans No. 253 and 2 · 10 ⁶ in 1 cm ³ distilled water for Mucor racemosus No. 195.

Another group of rabbits in the amount of 6 goals, which were injected with saline, was a control.

After 5 days, all animals of the control group died, and 4 rabbits survived in the experimental group. Efficiency was 66.7%.

A vaccine with a different content of components was prepared as described in example 1.

Thus, the claimed vaccine is effective, immunogenic, does not have allergenicity, acts simultaneously against candidiasis and mucorosis.

Table 2.
The effectiveness of the associated vaccine Live group number Composition of the complex vaccine Content% Vaccine Administration Fell Survived goals % goals % one. - antigen based on a strain of Candida albicans No. 253;
- antigen based on strain Mucor racemosus No. 195 fifty
fifty four 66.7 2 33.3 2. - antigen based on a strain of Candida albicans No. 253;
- antigen based on strain Mucor racemosus No. 195 45
55 3 50,0 3 50,0 3. - antigen based on a strain of Candida albicans No. 253;
- antigen based on strain Mucor racemosus No. 195 40
60 2 33.3 four 66.7 four. - antigen based on a strain of Candida albicans No. 253;
- antigen based on strain Mucor racemosus No. 195 55
45 four 66.7 2 33.3 5. - antigen based on a strain of Candida albicans No. 253;
- antigen based on strain Mucor racemosus No. 195 35
65 3 50,0 3 50,0

Claims (1)

A vaccine against rabbit visceral mycoses containing a soluble antigen from the strain of the fungus Mucor racemosus No. 195 in the form of endotoxin of its cell wall and cytoplasm, characterized in that it additionally contains a soluble antigen from the strain of the fungus Candida albicans No. 253 in the form of its cell wall and cytoplasm culturing 28-36 hours, and the concentration of fungus cells 3 × 10 ⁸ -4 10 ⁸ in 1 cm ³ of distilled water, wherein the vaccine contains cell wall and endotoxin cytoplasm fungus Mucor racemosus №195 cultivation period with 48-56 hours and cell density g iba 7 × 10 ⁶ -13 x 10 ⁶ in 1 cm ³ of distilled water at the following ratio, wt.%:
Mushroom strain antigen Mucor racemosus No. 195 in the form endotoxin of its cell wall and cytoplasm with cell concentration mushroom 7 · 10 ⁶ -13 · 10 ⁶ in 1 cm ³ distilled water 55-65 Mushroom strain antigen Candida albicans No. 253 in the form cell wall and cytoplasm with cell concentration mushroom 3 · 10 ⁸ -4 · 10 ⁸ in 1 cm ³ distilled water 35-45