RU2281507C2 - Method for evaluating toxicity of bacterial antigens - Google Patents

Abstract

FIELD: microbiology, immunology.

SUBSTANCE: the culture of Paramecium caudatum infusorias should be supplemented with a tested antigen in different dilutions and kept for 3 min at a room temperature to count the number of contractions of excretory vacuoles/min in 6-7 infusorias, detect toxic impact of bacterial antigen according to suppression degree of infusorial excretory function, and toxic concentration is stated upon at antigen concentration that decreases the number of contractions of infusorial excretory vacuoles against the control by 50%. Thus, a highly sensitive and rapid method for evaluating toxicity of bacterial antigens has been successfully elaborated.

EFFECT: higher accuracy of evaluation.

5 dwg, 1 ex, 2 tbl

Description

The invention relates to medicine, in particular to microbiology and immunology, and may find application in the development of new chemical vaccines based on bacterial antigens. To date, practical health care has no vaccines against some especially dangerous infections, including glanders and melioidosis. Melioid infection in modern conditions has a tendency to spread far beyond the endemic foci due to the expansion of economic and cultural international relations and the strengthening of population migration processes. In this regard, the problem of creating a chemical vaccine against melioid infection remains very urgent.

One of the important and necessary steps in the selection of bacterial antigens as possible components of a chemical vaccine is the study of their toxic properties.

The toxicity of bacterial antigens is studied both in laboratory animals in vivo and in vitro on models of various cells, in particular, on animal macrophage cultures and cultures of transplantable cell lines (1). A method for assessing toxicity in unicellular organisms - ciliates is also used in medical and hygienic studies, it is sufficiently simple and can be used in any sanitary and bacteriological laboratory. Ciliates, being a cell and an organism at the same time, make it possible to evaluate a variety of effects both at the cellular and at a higher organism level.

A method for determining toxicity on a culture of Tetrachymena pyriformis ciliates is described by the degree of growth inhibition of the culture under the influence of the test substance (2). However, this method is quite laborious, associated with the need for daily cultivation and multiple measurements of the density of the culture when counting in the Goryaev’s chamber, as well as with the need for formalin treatment of ciliates. At the same time, it remains unclear due to what mechanisms the number of ciliates decreases: due to their partial death or due to inhibition of the reproduction process or for other reasons.

The closest analogue is work (3), in which the criterion for the death of ciliates is used to assess the toxicity of substances, however, this method is not sensitive enough, since it does not allow determining toxic doses of substances that disrupt the vital functions of cells without irreversible consequences, which inevitably lead to their death.

The purpose of the invention is to increase the sensitivity, simplification and acceleration of the method for assessing the toxicity of bacterial antigens.

This goal is achieved by registering functional changes in ciliates that occur long before gross morphological disorders leading to cell death. As a criterion for assessing the toxicity of antigens, it is proposed to use a 50% reduction in the number of contractions of excretory (contractile) vacuoles of Paramecium caudatum ciliates when a test antigen is added to their culture.

The ciliates of Paramecium caudatum have two contractile (excretory) vacuoles - one in the front and the other in the back of the body (Fig. 1). Vacuoles alternately contract as they are filled with liquid with metabolic products and release the contents into the environment through a special hole in the cell wall - the excretory pore. The duration of the interval between two contractions of vacuoles (pulsation frequency) depends on the ambient temperature (at a temperature of 16 ° C it is 3-4 in min) (4). When a toxic substance is added to the medium, it immediately enters the ciliates, as absorption occurs over the entire surface of her body. Toxic substances entering the cell disrupt its vital activity, which affects the excretory system, the number of contractions of excretory vacuoles decreases until their function is completely blocked. With the complete cessation of contractions, the vacuoles are overflowed with metabolic products, their volume increases significantly (Fig. 2). The next stage of the detrimental effect of the toxin on the ciliates at its high concentration in the medium is gross morphological changes in the cell wall of the ciliates in the form of multiple round outgrowths ("vacuolization" of walls), which are then torn and the contents of the cell (endoplasm) enter the environment (Figs. 3, 4).

A method is proposed for assessing the toxicity of bacterial antigens at the physiological level - by reducing the number of reductions in excretory vacuoles by 50%, when morphological changes in the cell do not yet occur.

The methodology of the experiment to determine the toxicity of bacterial antigens

A solution of bacterial antigen (in physiological saline) in the initial concentration is sequentially diluted in the wells of the polystyrene plate. Toxicity testing begins with the lowest antigen concentration. To do this, 100 μl of culture medium with infusoria is added to a separate well of the plates, 100 μl of antigen solution is added and left for 3 min at room temperature. In a control well to 100 μl of medium with ciliates add 100 μl of physical. solution. After 3 min, 25 μl of the contents of the experimental and control wells are placed on a glass slide, several cotton wool fibers are added to limit the movements of the ciliates, the coverslip is covered from above and the preparations are microscopically examined under a light microscope using blue filters or a phase contrast device. Count the number of contractions of excretory vacuoles per minute in 6-7 individuals. At a water temperature of 28-30 ° C without the addition of a toxic substance, the number of reductions was usually 7-8 per min. In experimental samples, depending on the degree of antigen toxicity, the number of reductions decreased until they were completely absent. Such a concentration of antigen was taken as the toxic dose, which depressed the excretory function of the ciliates by 50%, i.e., the number of vacuole contractions was 3-4 per min (CTE 50). The nutrient medium for ciliates Paramecium caudatum is an infusion of meadow hay (10 g per 1 liter of water), which is boiled for 15 minutes, during which all protozoa and their cysts die, but hay stick spores remain. After 2-3 days, bacteria necessary for the nutrition of Paramecium develop from the spores. A culture of ciliates is introduced into the prepared nutrient solution, which is further cultivated at room temperature and in intense sunlight.

Example 1

The following bacterial antigens were used to determine toxic properties:

1 - water-salt extract (SEE) of bacteria of the causative agent of melioidosis - Burkholderia pseudomallei C-141;

2 - BCE of the bacteria of the pathogen glanders - Burkholderia mallei B-120;

3 - FEV of non-pathogenic burkholderia-Burkholderia thailandensis;

4 - ultrasonic (US) disintegrate of bacteria of the plague pathogen - Yersinia pestis 358/80;

5 - ultrasonic disintegration of bacteria of the pathogen of glanders B.mallei C-5;

6 - V fraction according to Baker, obtained by fractionation of ammonium sulfate water-salt extract of bacteria of the plague pathogen Y. pestis 231;

7 - cholera toxin from the causative agent of cholera - Vibrio cholerae (Serva);

8 - actinomycin C (Serva).

Actinomycin C was used as a control drug, known for its high toxicity, cytopathogenic and immunosuppressive effects. The study of each antigen was carried out in 6 replicates. Statistical analysis of the results was carried out with the determination of average values and confidence intervals for a confidence level of 95% (5).

We have shown that doses of bacterial antigens that cause "vacuolization" of the cell wall and destruction of ciliates (DCL) are 8-26 times higher than doses that inhibit their excretory function by 50% (CTE 50) (Table 1, Fig. 5).

Table 1
Parameters of the toxic effect of bacterial antigens on the culture of ciliates according to the criteria of inhibition of excretory function and cell death №№p / p A drug Toxic effect, μg / ml (M ± m) Inhibition of excretory. functions (CTE 50) Cell death (DCL) DCl / CTE50 ratio one 2 3 four 5 one, VSE B. pseudomallei S-141 3.02 ± 0.96 33.8 ± 4.1 10.5 2. VSE B.mallei B-120 2.14 ± 0.59 27.1 ± 3.32 12.6 3. VSE B.thailandensis 3.82 ± 1.1 46.5 ± 4.68 12.1 four. Ultrasound disintegrate Y.pestis 358/80 6.65 ± 1.34 58.5 ± 5.09 8.7 5. Ultrasound disintegrate B.mallei Ts-5 2.91 ± 0.63 28.12 ± 3.12 9.7 6. V fraction according to Baker from Y. pestis 231 3.82 ± 0.94 70.0 ± 5.84 18.3 7. Cholera toxin 0.36 ± 0.08 7.02 ± 1.63 19.5 8. Actinomycin C 0.33 ± 0.06 8.58 ± 1.76 26.0

We conducted a comparative study of the toxicity of bacterial antigens on different types of cells: mouse fibroblasts L-929, ovarian tumor cells of the Chinese hamster CHO K-1, peritoneal macrophages (PM) of BALB / C mice and ciliates Paramecium caudatum. The toxicity on L-929, CHO K-1, and PM cells was evaluated by the usual method after 24 hours of contact with the antigen by the death of 50% of the cells in the wells of the polystyrene plate (1), toxicity on the ciliates - by inhibition of 50% of their excretory function.

The summary results of several experiments are presented in Table 2.

table 2
Comparative toxicity of bacterial antigens for different types of cells №№
p / p A drug Toxicity, μg / ml (M ± m) СНО К-1 L-929 PM R. caudatum one. FEE B.pseudomallei C-141 2.76 ± 0.71 3.52 ± 0.86 8.36 ± 1.62 3.02 ± 0.96 2. FEE B.mallei B-120 2.83 ± 0.54 6.4 ± 1.68 18.06 ± 2.44 2.14 ± 0.59 3. FEE B.thailandensis 3.83 ± 1.56 4.46 ± 1.8 9.0 ± 1.9 3.82 ± 1.1 four. Ultrasound disintegrate Y.pestis 358/80 12.9 ± 1.94 9.7 ± 2.3 22.7 ± 3.1 6.65 ± 1.34 5. Ultrasound disintegrate B.mallei Ts-5 3.36 ± 1.13 6.49 ± 2.26 14.56 ± 2.31 2.91 ± 0.63 6. V fraction according to Baker from Y. pestis 231 3.96 ± 1.2 6.4 ± 1.82 18.06 ± 2.32 3.82 ± 0.94 7. Cholera toxin 0.22 ± 0.04 0.68 ± 0.11 0.32 ± 0.04 0.36 ± 0.08 8. Actinomycin C 0.17 ± 0.03 0.91 ± 0.13 0.40 ± 0.07 0.33 ± 0.06

As can be seen from Table 2, the method for determining toxicity by inhibition of excretory function at the ciliates is comparable in sensitivity to testing on cells of the tumor lines L-929 and CHO K-1, however it compares favorably with speed and simplicity. In addition, work with transplantable cell lines is impossible without special equipment and reagents (CO ₂ - incubator, laminar cabinet, special nutrient media and sterile dishes for cultivation).

The results show that the proposed method for assessing the toxicity of bacterial antigens at the ciliates of Paramecium caudatum is simple, able to obtain a quick response and allows toxicity to be assessed at a physiological level.

BIBLIOGRAPHY

1. Salnikova O.I. Testing and study of cholera vibrio toxins in the culture of monolayer cells / Diss.kand.biol.nauk, Rostov-on-Don, 1994.

2. Etlin S. N., Lakhonina G. M., Irlina I. S., Popova L. A., Malygin S. A. (The study of the relationship between the toxicity parameters of chemicals for ciliates Tetrahymena pyriformis and animals // J. Hygiene and sanitation, 1987, No. 9, S.80-82.

3. Tsikunib AD. Determination of endogenous intoxication by toxicuria using Tetrahymena pyriformis // Klin. laboratory. Diagnostics, 2001, No. 6, S.50-52.

4. Zelikman A.L. / Workshop on the zoology of invertebrates // M., "Higher School", 1969, S. 31.

5. Ashmarin I.P., Vorobyov A.N. Statistical methods in microbiological research // L., 1962, S.10-21.

Claims (1)

A method for determining the toxic concentration of a bacterial antigen. characterized in that the studied antigen is introduced into the culture of ciliates Paramecium caudatum in various dilutions, kept for three minutes at room temperature, the number of contractions of excretory vacuoles for 1 min is calculated for 6-7 ciliates, the toxic effect of the bacterial antigen is determined by the degree of inhibition of the excretory function of ciliates and for the toxic concentration, the antigen concentration is taken, which reduces by 50% the number of reductions in the excretory vacuoles of the ciliates compared to the control.

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