RU2344169C1 - Viability preserving medium for clinical isolates of pathogenic treponema palladium - Google Patents

Abstract

FIELD: chemistry, biochemistry.

SUBSTANCE: invention refers to viability preserving medium for clinical isolates of Treponema Palladium. Medium by the invention contains 0.6-0.75 ml of physiologic saline, 0.09-0.11 ml of glycerine and 0.03-0.06 ml of human tissue-like liquid (reiz-serum) containing infectious agent of syphilis and produced by gentle stimulation of erosive and ulcerative clinical surface in patient suffering from syphilis. Produced medium is carefully agitated, controlled for pathogenic Treponema Palladium, immediately frozen and stored at temperature minus 70…80°C. Medium by the invention is guaranteed to reserve viability and virulence (pathogenic power) of wild strains of infectious agent of syphilis.

EFFECT: generation of conditions for genetic researches aimed at studying of Treponema Palladium variability escaping production of laboratory strain.

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Description

The invention relates to medical microbiology and can be used to monitor the biological and genetic properties of the syphilis pathogen Treponema pallidum (pathogenic pale treponema), as well as to maintain the viability of "wild strains" of pathogenic pale treponema and to develop methods for serodiagnosis of syphilis using corpuscular antigens.

Experimental studies have established that pale treponema during cultivation on artificial nutrient media lost the property of pathogenicity [1, 2, 3].

It is known to use laboratory animals (biological models) to preserve the clinical isolates of pathogenic pale treponema taken from people with syphilis. So, by infection of various laboratory animals (primates, rabbits, guinea pigs), laboratory strains of pathogenic pale treponema (Nichols, Budapest, 1 Irkutsk, VIII and XII CCVI and others) were obtained and described in detail [3, 4]. The viability of these laboratory strains was maintained by sequentially inoculating them in laboratory animals. Currently, in the Russian Federation in vivariums at large clinical diagnostic laboratories of dermatovenereological dispensaries, there is only one strain of pathogenic pale treponema - the Nichols strain.

Known environment for maintaining the viability of pathogenic Treponema pallidum, the composition of which includes physiological saline containing tissue extract of the testes infected with syphilis rabbit and glycerin, added at the rate of 15% to the volume of medium. The viability of the pathogenic Treponema pallidum in the preservation medium is ensured by rapid freezing and subsequent storage at low temperatures (minus 70 ... 80 ° C) [5]. This medium for the preservation of pathogenic pale treponema was chosen as the closest analogue.

However, to use the known medium for preserving pathogenic pale treponema, it is necessary to initially obtain a culture (laboratory strain) of pathogenic pale treponema adapted to the living conditions in the laboratory animal (rabbit) organism and only then it is possible to guarantee the viability of the causative agent suspension obtained from rabbit orchitis in accordance with the above method . The procedure for obtaining a new laboratory strain is time-consuming, since the process of adapting a microorganism to the conditions of existence of another biological species in the body takes a long time. This procedure is expensive and, in some cases, not feasible for a number of organizational reasons (the need for vivarium, as well as the possibility of prompt infection of a rabbit with material from a patient with syphilis) [3, 4]. At the same time, there is a lack of stability in the results of obtaining new laboratory strains of pathogenic pale treponema and the impossibility of re-reproducing the strain in the case of a laboratory animal due to the patient having syphilis (the source of the clinical isolate of “wild” pathogenic pale treponema), in accordance with current standards, must begin specific treatment no later than 24 hours after diagnosis.

An object of the invention is to develop an environment for the long-term preservation of the viability of clinical isolates of “wild” pathogenic pale treponema obtained directly from people with syphilis, bypassing the stage of obtaining a laboratory strain.

This task is achieved due to the fact that, as a suspension of pathogenic pale treponems, a native interstitial fluid (raitz-serum) is used that contains the causative agent of syphilis, obtained by irritating the surface of erosive or ulcerative morphological elements on the skin and mucous membranes of a person with syphilis. The interstitial fluid obtained by the above method is collected in fractional portions and in an amount of at least 0.03-0.06 ml, subject to sterility conditions, transferred to eppendorf tubes containing 0.6-0.75 ml of physiological saline and 0.09-0 , 11 ml of glycerol, mix, control the content of the pathogen of syphilis in the resulting medium, freeze and store at a temperature of minus 70 ... 80 ° C.

The technical result of the invention is to simplify the procedure for maintaining the viability of clinical isolates of pathogenic pale treponema obtained directly from patients, since this eliminates the long stage of obtaining a laboratory strain of T. pallidum in laboratory animals.

Benefits of the proposed conservation environment:

- the use of the preservation medium allows you to maintain the viability of "wild" strains of pale treponema with the preservation of its pathogenic (virulent) properties;

- to obtain an environment for the preservation of clinical isolates of pathogenic pale treponema no expensive reagents are required;

- the use of the proposed environment for the preservation of clinical isolates of pathogenic pale treponema reduces the direct economic costs associated with the acquisition and maintenance of laboratory animals used for primary infection or passages of infectious material.

In addition, the developed medium does not contain additional ingredients that can cause a biological reaction, as a result of which the clinical isolates of pale treponema can be used for research immediately after defrosting (thawing), without washing or other additional processing. The possibility of preparing several separate aliquots of the preservation medium with pathogenic pale treponema allows you to arbitrarily control the execution time of various types of work with the obtained clinical isolate of pale treponema (molecular methods for studying genetic markers of the syphilis pathogen, their adaptation to the living conditions in the body of a laboratory animal in order to obtain new laboratory strains, restoration of a lost laboratory strain of pathogenic pale treponema).

The invention is as follows.

The surface of the erosive (ulcerative) element on the skin or mucous membranes of a patient with syphilis is cleaned with a sterile swab moistened with sterile saline. Then, with stroking movements of Volkman's sterile plastic spoon, they lightly irritate the surface of the element, avoiding damage to small blood vessels. These actions cause a flow of interstitial fluid from the depth of the inflamed tissue to the surface. The interstitial fluid (raitz-serum) is collected with a Volkman plastic spoon containing 0.010-0.015 ml. Repeated actions transfer 0.03-0.06 ml (5-10 doses) of interstitial fluid into a sterile eppendorf tube containing 0.6-0.75 ml of physiological saline and 0.09-0.11 ml of glycerol. The medium is thoroughly mixed, a preparation is prepared for microscopy of pale treponema in a dark field of view, the tube with the medium and the pathogen of syphilis is closed with a stopper.

The content of pathogenic pale treponema in the prepared medium is controlled by microscopy of the native preparation on a glass slide covered with a coverslip in a dark field of view (eyepiece - x7, lens - x40, binocular nozzle - x1.5). The prepared preparation of the medium should contain mobile pale treponemas with typical morphology and characteristic types of movements. The number of treponemas should be at least 2-5 in the field of view, which corresponds to a concentration of 2-5 · 10 ⁵ microorganisms in 1 mm ³ . At the same time, the concentration of syphilis pathogens in the medium does not affect the preservation of the viability and virulence of the clinical isolate of pathogenic pale treponem. A study of the prepared medium is performed to control the presence and visual identification of typical pale treponemas.

To maintain the viability of the clinical isolate of pathogenic pale treponema, obtained together with the Ritz-serum, the in vitro medium is quickly frozen and subjected to storage at a temperature of minus 70 ... 80 ° С. The shelf life of the virulent properties of the clinical isolates of pathogenic pale treponema using the developed medium according to experimental data is at least 12 months.

The effectiveness of the developed environment is illustrated by examples where a biological model of a laboratory animal (rabbit) was used to assess the viability and virulence (pathogenicity) of clinical isolates of pathogenic pale treponema:

Example 1

January 31, 2006, from a patient M. with secondary syphilis by delicate irritation of the surface of an erosive chancre in the area of the coronary sulcus, a suspension of pale treponemas in the interstitial fluid was obtained. Following aseptic rules, it was collected with a Volkman spoon and transferred in an amount of about 0.05 ml to eppendorf tubes containing 0.6 ml of sterile physiological saline and 0.09 ml of sterile glycerol. In total, 5 duplicates of the preservation medium with a clinical isolate of the syphilis pathogen were prepared. After thorough mixing, control native medium preparations were prepared, and test tubes with the medium were immediately frozen and stored at minus 70 ... 80 ° С.

In the native preparation under microscopy in a dark field (eyepiece - x7, lens - x40, binocular nozzle - x1.5), mobile pathogenic pale treponemas with typical morphology and characteristic types of movements in the amount of 6 to 10 in the field of view were found, which corresponded to the content 6 · 10 ⁵ -1 · 10 ⁶ microorganisms in 1 mm ³ .

05/18/2006, 3.5 months after placing in a low-temperature refrigerator a preservation medium containing pathogenic pale treponema (obtained from patient M.), two tubes were thawed at room temperature, and their contents were used to infect a healthy rabbit (by injection with a syringe 0.7 ml of clinical material in a preservation medium inside each testicle).

When observing a rabbit, no clinical signs of syphilitic infection were established. With rabbit blood serum taken on day 22 after infection, weakly positive test results were observed in RPR (++) and RIPA (+ / ++ at a dilution of 1:80 and 1: 160). In blood serum samples taken 32-94 days after infection, an increase in the positivity of responses in the reactions was noted: RPR (++++, titer 1: 8-64) and RPGA (++++, titer 1: 1280-20960 )

During subsequent transplantation (passage) of organs (liver, kidneys, testicles, spleen) from a primary infected rabbit to a healthy laboratory animal, the development of a latent form of syphilis was observed, confirmed by the development of a humoral immune response in the form of production of reagin (defined in RPR) and treponemospecific (determined in RPGA) antibodies in high titer.

Example 2

December 20, 2006, 10.5 months after placing in a low-temperature refrigerator a storage medium containing pathogenic pale treponema obtained from patient M. in January 2006 and stored at a temperature of minus 70 ... 80 ° C, two tubes with a storage medium were subjected thawing at room temperature and their contents in the amount of 0.73 ml with a syringe was introduced into each testicle in a healthy rabbit.

When observing a laboratory animal for 80 days, no clinical signs of syphilitic infection were observed. With rabbit blood serum taken on day 25 after infection, positive results were obtained by examining it in RPR (+++) and RPGA (+++ at a dilution of 1: 160). In serum samples taken at a later follow-up date - 65-80 days after infection, an increase in the level of antibodies in serological reactions was also noted: RPR (++++, titer 1: 16-64) and RPHA (+++ +, titer 1: 1280-5120).

The passage of pieces of organs (liver, kidneys, testicles, spleen) from a primary infected rabbit to a healthy laboratory animal, performed 80 days after infection, demonstrated the development of a humoral immune response in response to the introduction of an infectious agent in the form of production of reagin and treponemospecific antibodies tested using RPR , RIF _c , and RPGA.

Thus, the above examples 1 and 2 demonstrate:

- successful receipt of a clinical isolate of pathogenic pale treponema from a patient with syphilis (confirmed by microscopy of control preparations);

- the possibility of long-term continuous storage of the clinical isolate of pathogenic pale treponema in the developed storage medium under low temperature conditions (minus 70 ... 80 ° C) for 3.5 and 10.5 months without loss of virulence (table);

- confirmation of the viability and pathogenic properties of clinical isolates of pathogenic pale treponema, preserved in the developed environment, carried out by experimental infection of laboratory animals. The development of a latent form of syphilitic infection in rabbits was established on the basis of the appearance of a humoral immune response in the terms typical for this method of infection. The fact that laboratory animals were infected with syphilis, and not their passive immunization with non-viable biological material, was confirmed by transplanting pieces of organs from primarily infected rabbits to rabbits of the first passage.

Table
The results of the study of the viability and pathogenicity of clinical isolates of pathogenic Treponema pallidum depending on the shelf life Learning Properties Shelf life of clinical isolates of pathogenic Treponema pallidum 0.5 month 3.5 months 6 months 10.5 months 12 months Viability and pathogenicity Saved Saved (example 1) Saved Saved (example 2) Saved

Information sources

1. D.K. Zabolotny. To the question of spirochete cultures. / / Selected works. Volume 2. Cholera. Syphilis. Epidemiological and other works. - Kiev: Publishing House of the Academy of Sciences of the Ukrainian SSR, 1951. - P.186-188.

2. R.R. Geltzer. Growing pale spirochetes and the importance of these cultures. // Collection of scientific works of the Stavropol Institute of Epidemiol. and microbiol., 1947. - Issue 1. - P.83.

3. N.M. Ovchinnikov. Experimental syphilis. - M .: Medgiz, 1955 .-- 388 p.

4. N.M. Ovchinnikov. Experimental rabbit syphilis.// Methods of experimental chemotherapy (Practical Guide) ./ Ed. G.P. Pershina. Vol. 2. - M .: Medicine, 1971. - P.89-91.

5. Patent RU No. 2292388 A1. A method for maintaining the viability of a pathogenic pale treponemal strain. A.A. Kubanova, S.V. Rotanov, N.V. Frigo, T.I. Milonova. / FGU TsNIKVI Roszdrav. - Bulletin "Inventions, utility models" from 01/27/07. - Number 3.

Claims (1)

An environment for maintaining the viability of clinical isolates of pathogenic Treponema pallidum (pale treponema) containing physiological saline, glycerin and a suspension of pathogenic pale treponema, characterized in that it contains interstitial fluid obtained from a pathogenic pale treponema obtained directly from the surface of erosive elements or ulcers a person with syphilis of a person (raitz-serum), with the following quantitative ratio of components, ml:
saline 0.6-0.75 glycerol 0.09-0.11 interstitial fluid 0.03-0.06