RU2332460C1 - Method of detection of epidemiologically relevant cholera vibrions vibrio eltor and vibrio cholerae o139 by their adhesion properties - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: method involves using II human blood type as a substrate for attachment of cholera vibrions. The blood undergoes defibrination, triple scouring by centrifugal separation at 3000 rpm. for 15 minutes together with 0.9% NaCl pH 7.2. Obtained erythrocytes are dissolved in physiological solution at a ratio of 1:8, then a slide test is set with one drop of 1 billionth suspension of cholera vibrion agar culture and one drop of 8% erythrocytes. Smears are placed for 20 minutes to a humidity chamber and incubated there at 37°C, with further preparation of samples for microscopic examination. Differentiation is performed by the reaction results: if average adhesion index AAI is over 1.5, then the cholera vibrion strain tested is toxigenic and epidemiologically relevant, and if AAI is less than 1.5, then erythrocytes are lysated, coloured red, and the strain is atoxigenic, hemolytic and epidemiologically irrelevant.

EFFECT: application in laboratory diagnostics for express analysis of cholera vibrions for detection of epidemiologically relevant, toxic and atoxigenic strains.

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Description

The present invention relates to medical microbiology and can be used in laboratory diagnostics for the rapid study of strains of cholera vibrios Vibrio eltor and Vibrio cholerae O139 (Bengal).

One of the main questions of epidemiological surveillance for cholera is the assessment of the epidemic significance of secreted cholera vibrio cultures existing in the form of toxigenic non-hemolytic strains that are detected on Greig's sheep erythrocytes and determination of the cholera toxin gene in the polymerase chain reaction (PCR) of cholerae sucker rabbits. PCR and determination of cholerogenicity in animals are laborious, expensive and not available to all laboratories. In this connection, the search for new simple express methods for the detection of toxigenic strains in vitro is an urgent problem.

A known method for determining the adhesive ability of vibrios using red blood cells (see S. G. Sappo, L. Ya. Urbanovich, BC Wheelchairs "Using red blood cells to detect the adhesive ability of vibrios" collection "Modern aspects of the prevention of zoonotic infections", Ministry of Health of the USSR, Irkutsk, 1984, p. 126-127), consisting in the use of rabbit erythrocytes, for this, smears were prepared from a suspension of the test culture of vibrio and red blood cells and stained according to Romanovsky-Giemsa and durable green - with dye azuro m II, then for each strain the “adhering” vibrios were counted on 100 red blood cells and the average number of “adhering” vibrios per one erythrocyte was determined.

However, despite the fact that the indicators make it possible to reliably determine the virulence of the strains by their adhesive ability, they do not allow differentiating the strains by their epidemic significance. In addition, the process itself is time-consuming and expensive, as it requires the maintenance of animals

For the prototype, a method was selected for determining the adhesive ability of cholera vibrios (see ed. St. USSR No. 1306107, class C12N 1/00, C12Q 1/02, Bull. 24, 06/30/90), using rabbit blood as a substrate, a suspension of which in a volume of 0.5-1% is incubated with the studied culture and taken in equal volumes for 25-40 minutes.

The disadvantage of the prototype is that the use of rabbit erythrocytes reveals the adhesive ability of vibrios with different virulence, and the latter may include strains that do not produce cholerogen, which does not reflect the correlation with the main factors of epidemic significance, namely, toxigenicity and hemolyticity, that is, rapid express displaying the presence of the cholera toxin gene and erythrocyte hemolysis in the absence of the latter.

The technical result of the proposed method is the rapid determination of the epidemic significance of strains of cholera vibrios Vibrio eltor and Vibrio cholerae O139 "Bengal", namely, the rapid detection of both toxigenic and hemolytic strains of cholera vibrios.

The specified technical result is achieved by the fact that in the known method for determining the toxigenicity of Vibrio eltor cholerae and Vibrio cholerae O139 cholera vibrios by their adhesive ability, including incubation of the test culture with subsequent preparation of microscopic preparations and determining the adhesive ability by the number of vibrios adhered to the substrate, and the latter is used The human blood group II, which is defibrinated, is washed three times, centrifuged at 3000 rpm for 15 minutes with 0.9% NaCl, pH 7.2, obtained erythrocytes are diluted with physiological saline in a ratio of 1: 8, then put the reaction on the glass, applying one drop of 1 billion suspension of the agar culture of cholera vibrios and one drop of 8% red blood cells, then put smears for 20 minutes in a moist chamber and incubate the latter at 37 ° С followed by preparation for microscopic studies, and differentiation is carried out according to the results of the reaction, if the average adhesion index is SPA> 1.5, then the studied strain of cholera vibrio is toxigenic - epidemiologically significant, and if and SPA <1.5, the red blood cells are lysed, have a red color, therefore, the strain is atoxygenic, hemolytic and has no epidemic significance.

The method is as follows.

Native human blood of group II is defibrified, centrifuged three times at 3000 rpm for 15 minutes, washed with 0.9% phosphate-buffered solution of pH 7.2. The supernatant is removed, the washed red blood cells are diluted with sterile saline in a ratio of 1: 8, namely 0.8 ml of red blood cells and 9.2 ml of NaCl.

Then, from a 18-hour agar culture of cholera vibrios, 1 billion suspension is prepared according to the turbidity standard of the CCO GISK them. Tarasevich.

1 billion suspension of culture and 1 drop of 8% red blood cells are applied dropwise to a glass slide. The glass is placed in a wet chamber for 20 minutes, then in a thermostat with a temperature of 37 ° C. After that, the smears are dried in air, fixed with 96 ° alcohol for 20 minutes, stained with Romanovsky-Giemsa methylene blue for 20 minutes, the glasses are washed three times with distilled water.

Adhesive ability is evaluated by the average adhesion index (SPA), which is determined using a light microscope based on the total number of vibrios attached to 100 red blood cells.

where SPA is the average number of vibrios attached to one red blood cell.

By the results of the reaction, differentiation is made. If the SPA is <1.5 and erythrocyte hemolysis takes place, which is visually fixed in the smear (because red blood cells are increased in size, have a red color and an irregular shape), then we conclude that the strain is non-toxic, hemolytic and does not have epidemic significance.

Example 1

The strain V. cholerae O139 Bengal 16131 (ctx ⁺ Hly ^- ) and 17781 (ctx ^- Hly ⁺ ) were used as the studied strain. As a substrate, red blood cells of I, II, III, IV human blood groups are used (see table 1).

From table 1 it is seen that the most demonstrative difference between toxigenic and non-toxigenic Bengal strains in human blood group II, as for strain 16131-SPA (0.5), and for strain 17781-SPA (1.1). In group III, there is no differentiation in adhesion. In groups I and IV, differentiation is less demonstrative than in group II blood. In addition, the Bengal hemolytic strain 17781 has not only the lowest SPA (1,1), but also erythrocyte lysis, which is fixed visually in the smear, where red blood cells are red, enlarged and irregular in shape. Thus, the strain "Bengal" 17781 - nontoxigenic and has no epidemic significance.

Strain "Bengal" 16131 has a cholera toxin gene, because the second blood group has the highest SPA (5.0), which is most demonstrative in differentiation. Erythrocyte hemolysis in the smear is not observed, i.e. red blood cells are colored blue, have a regular round shape and are not enlarged. Therefore, this strain has a cholera toxin gene of cholera vibrio, therefore, it poses an epidemic danger, as confirmed by PCR analysis.

Example 2

A culture of V. cholerae eltor 5879 (ctx ^- Hly ⁺ ) was used as the studied strain. As a substrate, red blood cells of I, II, III, IV human blood groups and strain V. cholerae eltor 14863 (ctx ^- Hly ⁺ ) are used (see table 2).

From table 2 we observe a demonstrative difference between toxigenic and non-toxigenic V. cholerae eltor strains also in human blood group II. In group IV, regular differentiation is not observed. It is not expressed in groups I and III. In blood group II, the hemolytic strain V. cholerae eltor 14863 has low CHA (0.6) and erythrocyte lysis, which is fixed visually in the form of lysed red erythrocytes, which confirms the absence of epidemic significance.

Strain V. cholerae eltor 5879, having the cholera toxin gene (ctx), has high adhesion (SPA = 7.3) and the absence of hemolysis. According to the results of adhesion, we can conclude that the strain is toxigenic and not hemolytic, and therefore epidemically significant, as confirmed by PCR analysis when the cholera toxin gene is detected.

Example 3

As the studied strains, cultures of V. cholerae eltor 5879, V. cholerae eltor 14863 (ctx ^- Hly ⁺ ), V. cholerae 0139 Bengal 16131 (ctx ⁺ Hly ^- ) and 17781 (ctx ^- Hly ⁺ ) were used. As a substrate, sheep and rabbit erythrocytes are used (see table 3).

Table 3 shows that adhesion to sheep and rabbit erythrocytes is not a differential sign between toxigenic and non-toxigenic strains of V. cholerae eltor and V. cholerae 0139 Bengal.

Thus, non-hemolytic strains, which are basically toxigenic, have a high adhesion index.

From the experiments carried out by specialists of the Rostov NIEPR it was found that the adhesion on rabbit and sheep erythrocytes does not reflect the toxigenicity and atoxigenicity of cholera vibrio strains. All strains adhere well regardless of the presence or absence of the cholera toxin gene and hemolyticity, have a high adhesion index, SPA> 2, but there is no natural differentiation between atoxigenic and toxigenic strains. The presence of the cholera toxin gene by the average adhesion can be determined on human I, II and III groups, however, on the II group, the difference in SPA between toxigenic and atoxigenic is most demonstrative (toxigenic - 5; atoxigenic - 0.6) and erythrocyte hemolysis is visible in the smear .

Thus, the present invention allows for the rapid determination of the epidemic significance of Vibrio eltor cholera vibrios and Vibrio cholerae O139 Bengal.

Table 1 Strain No. The presence of the ctx gene (in PCR) and hemolysis in the Greig sample SPA on human red blood cells and hemolysis I hemolysis II hemolysis III hemolysis IV hemolysis 16131 Bengal ctx ⁺ Hly ^- 2,3 - 5,0 - 1.8 - 2.6 - 17781 Bengal ctx ^- Hly ⁺ 1.9 - 1,1 + 1,2 - 1.4 -

table 2 Strain No. The presence of the ctx gene (in PCR) and hemolysis in the Greig sample SPA on human red blood cells and hemolysis I hemolysis II hemolysis III hemolysis IV hemolysis V. cholerae eltor 5879 ctx ⁺ Hly ^- 2,3 - 7.3 - 2 - 0.8 - V. cholerae eltor 14863 ctx ^- Hly ⁺ 1.37 - 0.6 + 1.75 - 1.3 -

Table 3 Strain No. The presence of the ctx gene (in PCR) and hemolysis in the Greig sample Red blood cell spa ram a rabbit V. cholerae eltor 5879 ctx ⁺ Hly ^- 4.8 3.8 V. cholerae eltor 14863 ctx ^- Hly + 1.38 four V. cholerae O139 Bengal 16131 ctx ⁺ Hly ^- four 2 V. cholerae O139 Bengal 17781 ctx ^- Hly + 3.6 3.4

Claims (1)

A method for detecting epidemiologically significant cholera vibrios V. cholerae eltor and V. cholerae O139, comprising incubating the test culture with subsequent preparation of microscopic preparations and determining the adhesive ability by the number of attached vibrios to the substrate, characterized in that human blood group II is used as a substrate, which defibrinate, wash three times, centrifuging at 3000 rpm for 15 min with 0.9% NaCl pH 7.2, the resulting red blood cells are diluted with saline in a ratio of 1: 8, then put the reaction on glass, applying one drop of 1 billion suspension of agar culture of cholera vibrios and one drop of 8% red blood cells, then put smears for 20 minutes in a moist chamber and incubate them at 37 ° C, followed by preparation for microscopic studies, according to the results of the reaction, differentiation is made, if the average adhesion index of SPA is> 1.5, then the studied strain of cholera vibrio is toxigenic - epidemiologically significant, and if SPA <1.5, red blood cells are lysed, have a red color, therefore, the strain is atoxygenic, hemolytic and has no epidemic significance.