RU2292388C1 - Method for retaining pathogenic treponema pallidum strain viability - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves producing pathogenic Treponema pallidum strain suspension from rabbit testis infected with Treponema pallidum. The testis is mechanically comminuted and extracted in isotonic sodium chloride solution. The suspension has native infected rabbit testis tissue extract. Then glycerol is added in the amount of 15% of total medium volume. The produced product is divided into aliquots of 0.75-1.5 ml and frozen immediately for storing it at -70 - -80°C in future after having stirred.

EFFECT: enhanced effectiveness in retaining infectious agent virulence.

Description

The invention relates to medicine, namely to microbiology, and can be used in an experiment to study the biological properties of the pathogen of syphilis, while maintaining and maintaining a strain of pathogenic pale treponema for industrial production, in specialized laboratories for serodiagnosis of syphilis using immobilization reactions and indirect immunofluorescence.

The causative agent of syphilis, pale treponema, was isolated in 1905, belongs to the species Treponema pallidum (Schaudin et Hoffman). In subsequent years, by infection of laboratory animals (primates, rabbits, guinea pigs, mice) with biological material from people with syphilis, strains of pathogenic pale treponema (Nichols, Budapest, 1 Irkutsk, VIII and XII CCVI and others) were described in detail. , the subsequent preservation of the strains was carried out by regular inoculations on laboratory animals.

Attempts to grow pale treponema on nutrient media under anaerobic conditions led to the production of a certain number of strains of cultural pale treponema (Stavropol strains 1-5,7-9, Reuters and others). When studying morphology and biology, certain differences were found between strains of pathogenic and cultured pale treponemas, differences in antigenic determinants were revealed even within these two groups of pale treponema. None of the cultures of pale treponema has pathogenicity for humans.

There are publications about the possibility of 1-3 times the passage of pathogenic pale treponema on cell cultures, but at the same time, there is a gradual loss of the ability to grow and multiply after one or two passages, which ultimately leads to the death of the strain.

As a laboratory animal for the preservation and inoculation of pathogenic pale treponema, the rabbit is most acceptable. Various methods have been developed for obtaining the suspension of the pathogen for experimental and research purposes (infection in the anterior chamber of the eye, intradermal inoculation in shaved areas on the back or scrotum, infection inside the testicle and others) (N.M. Ovchinnikov. Experimental syphilis. - M .: Medgiz , 1955. - P.388).

The main disadvantage of the method is the need for regular transplantation of the strain, taking into account the possibility of losing the strain, not only because of an unintentional violation of the transplantation technique, but also because of the death of the laboratory animal. For a more successful suspension with an abundant content of the pathogen, an additional administration of a suspension of hydrocortisone to rabbits is used, which leads to a decrease in the function of the mechanisms of humoral and cellular immunity of animals and can increase mortality under adverse conditions. These difficulties in preserving the strains lead to the need for a systematic duplication of the number of infected animals, maintaining many commercial ties with institutions that also preserve strains of pathogenic pale treponema.

A known method of maintaining the viability of a strain of pathogenic pale treponemas in the tissues of the testicle, obtained 7 days after infection of the rabbit, by freezing the testicle in its entirety at a temperature of minus 20 ° C (V.E. Pavlovskaya. The use of frozen antigen for the reaction of immobilization of pale treponemes. // Laboratory work. - 1964. - No. 11. - S.668-670). This method is selected for the prototype.

The main disadvantages of this method: the author noted the stable preservation of mobility of treponemes only for 1-2 weeks and a significant decrease in the number of pale treponemas in the material obtained from testicular tissues stored for 3-4 weeks. In addition, the author showed a marked decrease in the virulence of pale treponemas during storage: when the strain was inoculated with material from the testicle of an infected rabbit, which remained for 30 days at a temperature of minus 20 ° С, the formation of syphilitic orchitis increased to 20-30 days compared to 7 days incubation when using fresh, not subjected to freezing suspension of microorganisms. The lack of stability in the timing of syphilitic orchitis according to the described method does not allow to confidently plan experimental and laboratory studies, leads to unjustified material costs for the additional maintenance of healthy animals before the start of the experiment. Only a single use of the stored biological material is possible.

The objective of the invention is to increase the shelf life of strains of pathogenic pale treponema, reduce direct economic costs for the acquisition and maintenance of laboratory animals-understudies, create the ability to arbitrarily regulate (suspend) transplantation of strains in situations of an economic or epidemiological nature.

This task is achieved due to the fact that from a rabbit testicle infected with pale treponemas by mechanical grinding and extraction in an isotonic sodium chloride solution, a suspension of the pathogenic pale treponema strain is obtained, 15% glycerol of the total volume of the medium is added to it, after mixing, it is divided into small aliquots of 0 volume , 75-1.5 ml and immediately frozen for subsequent storage at a temperature of minus 70-80 ° C.

The addition of 15% glycerol as a cryoprotectant prevents the destruction of microorganisms, separation into small aliquots of 0.75-1.5 ml in volume makes it possible to achieve optimal freezing and thawing rates of the obtained product, and subsequent storage at a temperature of minus 70-80 ° C ensures the preservation of microorganism viability without loss of their virulence for at least a year (according to experimental studies).

The advantages of this method:

- during the uninterrupted operation of refrigeration equipment, the virulence of pathogenic pale treponema is guaranteed;

- a suspension of pathogenic pale treponema can be used to infect rabbits immediately after thawing (thawing) without additional washing or other processing, since the storage medium includes tissue components necessary for the vital activity of microorganisms, and does not contain materials or ingredients that can cause an incompatibility or rejection reaction from the side of the laboratory animal;

- the timing of the development of syphilitic orchitis when using a suspension of pathogenic pale treponema, preserved by the proposed method, does not differ from the development time after infection with a fresh suspension of the pathogen (7 ± 2 days);

- storage of suspension in the form of separate aliquots allows fractional use of the originally prepared biological material without compromising its properties and repeatedly reproducing the inoculations of the strain;

- the systematic use of this method reduces the direct costs associated with the acquisition and maintenance of laboratory animals-understudies (at least 2 times) or necessary for rabbit passages with a temporary cessation of transplantation of a strain of pale treponema.

The method is as follows:

From a rabbit testicle infected with pale treponemas by mechanical grinding and extraction in an isotonic sodium chloride solution, a suspension of a pathogenic pale treponema strain is obtained containing an extract of the native tissues of the infected rabbit's testicle, 15% glycerol of the total medium volume is added, mixed, divided into small aliquots of 0.75 volume -1.5 ml and immediately frozen for subsequent storage at a temperature of minus 70-80 ° C.

Example No. 1

Rabbit No. 8196 (passage 389) was euthanized on November 18, 2003. Two testicles were obtained from it under sterile conditions, each of which was freed from the shells and mechanically crushed by numerous cutting with surgical scissors. To the prepared mass, 5.0 ml of isotonic sterile sodium chloride solution was added and extraction was performed while swaying on the platform of the orbital shaker. Large elements of the tissue of the testicle of the rabbit were precipitated by centrifugation at a speed of up to 1000 rpm, the suspension of pathogenic pale treponem obtained under sterility conditions was transferred to a test tube with a measuring pipette, 15% of the total volume of sterile glycerol was added and mixed thoroughly. The suspension prepared for preservation was poured into aliquots in 1.0 ml Eppendorf tubes; the tubes were corked with lids and placed in a freezer with a temperature of minus 70-80 ° С.

After 2.5 months of storage (February 3, 2004), the contents of 2 tubes after thawing at room temperature were used to infect rabbit No. 8217 (1.0 ml inside the testicle on each side). 7 days after infection (February 10, 2004), the rabbit developed bilateral specific orchitis. The testes were removed, and according to the method described above, a suspension of pathogenic pale treponemas was prepared, which was used as an antigen to formulate the immobilization reaction of pale treponema (RIBT) and the indirect immunofluorescence (RIF) reaction, as well as to infect rabbit No. 8219, in which in turn, after 7 days the development of specific orchitis was observed on both sides.

Example No. 2

On March 23, 2004, from rabbit No. 8231 (passage 405), a suspension of pathogenic treponemal pale Nichols strain was prepared according to the standard method, 15% glycerol was added to it. After thorough mixing, the suspension was poured into 1.5 ml Eppendorf tubes, the tubes were closed with lids and placed in a freezer with a temperature of minus 70-80 ° С.

The suspension remained at a temperature of minus 70-80 ° C for 12.5 months and was used on April 12, 2005 to infect rabbit No. 8296. After thawing at room temperature and thoroughly mixing, the material was drawn into a syringe and 1.0 ml was introduced into the testis on each side. To reduce the rate of antibody production, the rabbit received two intramuscular injections of a suspension of hydrocortisone acetate in a single dose of 0.4 ml before infection and 4 days after it. On the 7th day after infection (04/19/2005), the rabbit was euthanized by air embolism, and a standard method was used to prepare a suspension of pathogenic pale treponema, which contained up to 400 pale treponemas in the field of view of the microscope (good suspension density). The material was used as an antigen for staging serological reactions (RIBT and RIF), as well as for the next passage on rabbit No. 8297, which in turn, after 7 days (04/26/2005), observed the development of specific orchitis on both sides.

Claims (1)

A method of preserving the viability of a pathogenic pale treponema strain, including infection of a rabbit testicle with a pathogenic pale treponema strain and freezing, characterized in that a suspension of pathogenic pale treponem containing an extract of native tissues of the infected rabbit testicle is obtained from an infected rabbit testicle by extraction with isotonic sodium chloride, 15 of the total suspension of glycerol, mix, divided into small aliquots of 0.75-1.5 ml, immediately frozen and stored at temperature re - (70-80) ° C.