RU2485182C1 - Method of indirect estimation of strains virulence of pathogenic burkholderia on grounds of cytopathogenicity - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: as an object for indirect estimation apparently healthy leaves of the plant Peireskia aculeate are used, on which sterile surface the notches are made, the infecting doses of the strains Burkholderia pseudomallei and Burkholderia mallei under study in different concentrations are applied, placed on a wet swab and incubated at 32°C for 24-48 h. The extent of their cytopathogenicity is evaluated visually according to the damage to the leaf blade - maceration, ulceration and blackening. Simultaneously the level of virulence of strains of pathogens for laboratory animals is determined, and the extent of cytopathogenicity for P. aculeata is correlated with virulence for laboratory animals.

EFFECT: invention provides high sensitivity, reproducibility, versatility, ease and accessibility of the study.

5 tbl, 4 ex

Description

The invention relates to medicine, and specifically to microbiology, and for assessing the virulence of strains of the causative agent of melioidosis and glanders.

Melioidosis and glanders are known to be particularly dangerous infections, and the pathogens Burkholderia pseudomallei and Burkholderia mallei cause serious illness in humans and many animal species. Many factors, such as exotoxin, thermostable lethal toxin, antigen 8, as well as extracellularly secreted enzymes like proteases, phospholipases, alkaline phosphatase, hemolysins, are important in the realization of the pathogenicity of these closely related types of microorganisms (1,3-5).

In genetic studies, it is often necessary to simultaneously and quickly study a large number of strains, select among them virulent and avirulent variants, and clone the analysis of the population according to the degree of pathogenicity.

A common method for determining the virulence of microorganisms is infection of sensitive laboratory animals with the calculation of LD ₅₀ . The closest analogue to the proposed method are the “Methodological recommendations for laboratory diagnosis of melioidosis”, which describes the corresponding technique (2). However, the use of animals is fraught with many difficulties and limitations: the duration of the experiment, the danger of contact with infected animals, the relatively high cost and complexity of their breeding and keeping.

The purpose of the invention is the development of a less expensive and accelerated method of indirectly assessing the virulence of strains of the causative agent of melioidosis and glanders in the plant model Peireskia aculeata (prickly Peireskia).

P.aculeata is an unpretentious houseplant from the cactus family, characterized by rapid growth, resistant to disease. Unlike other plant models, the P. aculeata life cycle is many years. When using it, it is not required to germinate the seeds, apply special nutritional mixtures. The leaves of this plant for a long time retain their original appearance in a humid chamber. The use of P. aculeata is characterized by low cost, since one leaf blade is enough to study one strain.

Plant preparation

At the initial stage, a young shoot having 5-6 internodes (cuttings) is cut obliquely with a sharp sterile knife above the kidney, the lower leaves are removed, put in clean water, into which activated carbon is added, after the roots appear, they are planted in the soil to a depth of one or two centimeter. A plant is used in the work for at least 1 year old, grown in room conditions, having a healthy appearance. Leaves with a length of 3.5-4.0 cm are selected, washed with distilled water, placed in a Petri dish on a cotton swab moistened with distilled water, bottom side up. All components (distilled water, Petri dish, cotton swab, scalpel) must be sterile.

Preparing a bacterial culture to infect plants and animals

Strains of B. pseudomallei and B. mallei are grown on nutrient agar (Difco) or on agar based on casein hydrolyzate for 48 h at 32 ° C. Suspensions of 1 × 10 ⁹ m.k./ml in 0.15 M NaCl are prepared. according to the standard sample turbidity LLC "Ormet" (SOP No. 1-98) and make ten-fold dilutions to 1 × 10 ¹ MK./ml For infection, all dilutions obtained are used.

Plant infection

The lower side of the leaf blade is treated with 70% ethanol, with a scalpel on both sides of the central vein, make 3-4 notches 2 cm long, then apply 1 standard bacteriological loop of a suspension of B. pseudomallei and B. mallei in 0.15 M NaCl in various concentrations, depending from the goals and objectives of the study. As a control, sterile 0.15 M NaCl is used. Cups with crops are incubated at 32 ° C. Infected samples are examined after 24 and 48 hours. The level of cytopathogenicity of the strains is assessed visually by the degree of damage to the leaf blade (maceration, ulceration and blackening of the leaf blade, table 1). In parallel, virulence for animals is determined.

Animal infection

For the study, golden hamsters weighing 75 g are used. Infection with cultures of B. pseudomallei and B. mallei is carried out subcutaneously in the area of the inner thigh, in doses of 1 × 10 ⁰ -1 × 10 ⁸ mk. (5 animals per dose) and take into account the dynamics of death in groups. Virulent properties are estimated by the number of dead animals, calculating LD ₅₀ according to Kerber. During the observation period (21 days) an autopsy is performed, the nature of the pathomorphological changes in the internal organs of the dead animals is assessed, bacterial cultures are isolated and identified.

The degree of cytopathogenicity of B. pseudomallei and B. mallei for P. aculeata is compared with the level of virulence for golden hamsters.

Thus, the proposed method consists in preliminary selection of apparently healthy P. aculeata leaves, preparation of ten-fold dilutions from 1 × 10 ⁹ to 1 × 10 ¹ m.k. / ml of suspensions of B. pseudomallei and B. mallei strains grown on Difco nutrient agar at 32 ° C for 48 hours, applying the resulting suspension from each infectious dose in the amount of one bacteriological loop to the sterile notches of the leaf plate, placing the leaves with the cultures on a sterile wet swab in a Petri dish and incubating at 32 ° C for 24-48 hours, determining level of cytopathogenicity according to art Degree of change in leaf blade: 4 degree, when maceration, ulceration and blackening of the leaf is wider after 48 hours after application, 3 degree when maceration, ulceration and blackening of the leaf within the application area, 2 degrees when maceration is absent, ulceration and blackening leaf blade within the area of application of the culture, culture growth is possible, 1 degree, when maceration is absent, ulceration and darkening of the plastic sheet along the notches, culture growth is possible, in the control - a sterile leaf with anesennymi notches culture without addition - no change lamina.

Example 1. A comparative analysis of the cytopathogenicity of strains of B. pseudomallei and B. mallei for P. aculeata

A sheet of P. aculeata is washed with distilled water, placed in a Petri dish on a cotton swab moistened with distilled water, the bottom side up, which is treated with 70% ethanol, with a scalpel on both sides of the central vessel, make 3-4 notches 2-3 cm long, Then, suspensions of B. pseudomallei and B. mallei are applied in a solution of 0.15 M NaCl in various concentrations from 1 × 10 ⁹ m.k. / ml, respectively, to the standard sample of turbidity of LLC Ormet (SOP No. 1-98) and lower in the amount of one standard bacteriological loop. Cups with crops are incubated at 32 ° C. Infected samples are viewed after 24 and 48 hours. The level of cytopathogenicity of the strains is assessed visually by the degree of damage to the leaf blade (maceration, ulceration and blackening of the leaf blade, table 1). The results are presented in table 2.

Example 2. Comparative analysis of the virulence of strains of B. pseudomallei and B. mallei for golden hamsters

The animals are infected with cultures of B. pseudomallei and B. mallei in doses of 1 × 10 ⁰ -1 × 10 ⁸ m.k. / ml (5 animals per dose) and the dynamics of death in the groups is taken into account. During the observation period (21 days) an autopsy is performed, the nature of the pathomorphological changes in the internal organs of the dead animals is assessed, bacterial cultures are isolated and identified. The results are presented in table 3.

Example 3. Comparative analysis of virulence and cytopathogenicity of strains of B. pseudomallei and B. mallei

The degree of cytopathogenicity for the plant model of P. aculeata correlates with the level of virulence of the studied strains for golden hamsters.

Strains of B. pseudomallei virulent for golden hamsters (LD ₅₀ 1 × 10 ^0-1 mk) give a 3-4 degree of damage to the leaf plate P. aculeata, strains with low virulence do not cause damage, with intermediate values of virulence ( LD ₅₀ 1 × 10 ^2-3 m.k.) - affect the leaf surface to 1-2 degrees.

In the case of B. mallei, characterized by lower enzymatic activity, a different pattern is observed. Virulent strains (LD ₅₀ 1 × 10 ^0-1 mk) give a 1-2 degree of damage to the leaf blade, with reduced virulence (LD ₅₀ 1 × 10 ⁵ mk) - do not cause damage.

Example 4. Determination of the level of cytopathogenicity of strain B. pseudomallei VPA ** (revertant)

P. aculeata leaves are placed in sterile Petri dishes on cotton swabs moistened with sterile distilled water. The lower side of the leaf blades is wiped with a 70% ethanol solution, incisions are made with a sterile scalpel on both sides of the central vein, and B.pseudomallei VPA ** culture suspension (revertant) is applied at a concentration of 1 × 10 ⁸ -10 ² m.c. / ml in volume one bacteriological loop. Incubation is 48 hours at 32 ° C. As a control, sterile 0.15 M NaCl is used. The cytopathogenicity of the strain is judged by the zone of maceration, ulceration and blackening of the leaf blade and the dose that caused these changes. The results are presented in table 5.

The data presented indicate a rather high sensitivity of the method and the possibility of using it in various ways: for an indirect assessment of virulence, the choice of animal infection dose for the final determination of virulence, a comparative analysis of the virulence of a large number of strains based on cytopathogenicity, selection of virulent strains and with reduced virulence, for more a thin analysis of changes in virulence of individual strains of B. pseudomallei and B. mallei.

Thus, the proposed method has high performance, sensitivity, reproducibility, versatility, simplicity and accessibility and can be used for a preliminary assessment of the virulence of strains of B. pseudomallei and B. mallei.

Literature

1. Melioidosis. Sat scientific Ed. N.G. Tikhonova. - Volgograd: Lower. - Volzh.kn.izd-vo, 1995 .-- 224 p.

2. MU 4.2.2787-10.4.2 Control methods. Biological and microbiological factors. Laboratory diagnosis of melioidosis. Guidelines (approved by Rospotrebnadzor on December 6, 2010).

3. Piven N.N., Smirnova V.I., Kapliev V., Pozolkova G.M., Khrapova N.P. The role of surface antigens of Pseudomonas pseudomallei in the pathogenesis of melioidosis // Journal of Microbiol. - 1991. - No. 10. - S.8-12.

4. Sap. SB.scientific. Ed. N.G. Tikhonova. - Volgograd: Lower. - Volzh. Prince Publishing House, 1995 .-- 128 p.

5. Dejsilbert S., Butraporn R., Chiewsilp D. High activity of acid phosphatase of Pseudomonas pseudomallei as a possible attribute relating to its pathogenesity // Jap.J. Med.Sci.Biol. - 1989. - vol. 42, No. 2. - p. 39-49.

Claims (1)

A method for indirectly assessing the virulence of pathogenic burkholderia strains based on cytopathogenicity, which includes preparing cell suspensions of the studied strains of Burkholderia pseudomallei and Burkholderia mallei of different concentrations, followed by their administration to laboratory animals, characterized in that Peratia leaves are used as an object for indirect virulence determination. in different concentrations, the infecting doses of the studied strains of B. pseudomallei and B. mallei, for which initially apparently healthy leaves are selected, ten tnye dilution of 1 × 10 ⁹ to 1 × ¹⁰ January microbial cells / ml suspensions strains of B. pseudomallei and B. mallei grown on Difco nutrient agar at 32 ° C for 48 hours, after which from each infection dose applied slurry in the volume of one standard bacteriological loop on previously applied notches on a sterile leaf plate, they are placed on a sterile wet swab in a Petri dish, incubated at 32 ° C for 24-48 hours, cytopathogenicity is judged by the degree of change of the leaf blade - 4th degree, when through 48 h maceration, ulceration and blackening of the leaf wider than the area of application of crops s, 3rd degree, when maceration, ulceration and blackening of the leaf blade within the zone of application of culture, 2nd degree, when maceration is absent, ulceration and blackening of the leaf blade within the zone of application of culture, culture growth is possible, 1st degree, when maceration is absent, ulceration and darkening of the leaf plasty along the incisions, culture growth is possible, the control of the experiment is a sterile sheet with incisions without the addition of bacteria on which there are no changes in the lamina, the degree of cytopathology Values for P. aculeata correlate with virulence for laboratory animals.