SU1751196A1 - Strain of bacteria versinia enterocolitica serovara 03, used as an antigen for yersiniosis serodiagnosis - Google Patents

Abstract

Use: medical microbiologists, diagnostics - use as a reference corpuscular antigen for the agglutination reaction (RA) for the purpose of serodiagnosis of yersiniosis. The essence of the invention: pure culture strain HISS No. 189 is grown on solid nutrient medium (MPA, Hottinger) at 37 ° C for 18-24h. Using an 85% sterile solution of sodium chloride, the bacteria are washed off the agar and washed twice in the same solution by centrifuging

Description

The invention relates to medical microbiology and is the original strain of the yersiniosis pathogen used as a reference particulate antigen for the agglutination reaction for the purpose of serodiagnosis of yersiniosis in diagnostic and scientific research.

Serodiagnosis yersiniosis. along with bacteriological examination, is the main method of laboratory diagnosis of this infection. In a number of cases, due to the impossibility of diagnosing yersiniosis on the basis of clinical signs and the ineffectiveness of the bacteriological method (later on the time of the disease, intracellular localization of the pathogen, etc.), serodiagnosis is practically the only method for confirming the diagnosis. The reactions used for serodiagnosis — the agglutination reaction (RA) and the passive hemagglutinitis reaction (RPHA) —are unequal, since in the first case, antibodies to the antigens of the whole cells of the causative agent are detected in the blood serum of patients,

about

Of these, lipopolysaccharide, fixed on the surface of erythrocytes (commercial erythrocyte diagnostics).

An antigen used in RA for serodiagnosis of yersiniosis should be a homogeneous suspension of microbial cells (live or inactivated) of a reference, better than local, strain with typical properties and the same antigenic structure, which is expressed during the stay of the pathogen in the human body. The latter is especially important, since for Yersinia, a temperature-dependent expression of a number of physiological characteristics, including many antigens, is characteristic.

As an antigen for RA, reference strains from international collections are used. One of these strains is Y. emerocolitica No. 134 (International Center for Yersini, Paris), which is used for serodiagnosis of diseases caused by Yersinii of serovar 03. Strain No. 134 is adopted as a prototype.

The use of a reference strain as an antigen for RA is known. Before preparing the antigen, he is tested for culture and morphological properties on nutrient agar, the colonies are selected in S-form, and microorganisms are grown on weakly alkaline agar (Hottinger or MPA) at 17-24 ° C for 24-36 hours.

To prepare the antigen, a suspension obtained after washing the agar culture directly (alive culture) or after processing the microbial suspension with formalin (culture killed) is used.

The disadvantage of using strain No. 134 is that to obtain a homogeneous cell suspension, the culture is grown at a temperature below 30 ° C, when the microbe has flagella and the H-antigen is expressed, and some of the surface antigens that are involved in the pathogenesis are not expressed. Their expression occurs at the temperature of the macroorganism (37 ° C). An increase in incubation temperature to 37 ° C leads to spontaneous flocculation in microbial suspension due to the appearance of hydrophobic components in the outer membrane of cells, which in turn is caused by the functioning in the cells of strain No. 134 of a virulence plasmid with a molecular weight of 40-48 MD. The formation of flakes in the control of the antigen is an obstacle for RA with a culture grown at 37 ° C.

The purpose of the invention is to obtain a strain of Y.enterocolltica serovar 03, which has

pronounced antigenic activity without signs of spontaneous agglutination, used as antigen for RA. The resulting strain Y.enterocolitica No.

955L deposited in the collection of the State Research Institute of Standardization and Control of Medical Biological Preparations. L.A. Tarasevich (N; 189, reference number 010 07/115).

Morphological and physiological characteristics.

Gram-negative rod-shaped cells 0.5x1.1 µm, motile at a temperature below 30 ° С due to peritrichous flagella; at 37 ° C are fixed, they do not form flagella. Dispute and capsules do not form. When cultivated on liquid media (MPV, Hottinger's broth,

0 environment Clarke), regardless of the temperature, form a uniform turbidity and a small compact sediment. On the environment Endo after 24 h at 37 ° C form colonies with a diameter of 0.2-0.3 mm, convex,

5 round with a smooth edge, colorless, smooth, transparent, pasty consistency; after 48 h - with a diameter of 1.5-2.0 mm, pink. Similar morphologies have colonies on Hottinger's agar. The strain does not possess the property of calcium dependence. Biochemical activity. Ferments with the formation of acid: glucose, sucrose, mannitol, maltose, glycerin, sorbitol, trehalose; does not ferment 5 digests lactose, rhamnose, salicin, esculin, xylose, dulcite. Possesses urease, catalase, ornithine decarboxylase. Does not form indole, hydrogen sulfide. Citrate does not recycle, phenylalanine does not deaminate. The test with methyl red is positive, the oxidase test is negative, Voges-Proskauer is positive at 26 ° C and negative at 37 ° C.

Antigenic properties.

5 Reacts agglutination with adsorbed diagnostic serum obtained by immunizing rabbits with Y.enterocoiltica serovar 03 reference strain from the international collection. It has a common enterobacterial antigen and group antigens. Spontaneous agglutination does not result.

5 Genetic features.

The strain does not contain the usual for Yersinia pathogenic plasmid virulence with mol. m, 40-48 MD, whose genes control the synthesis of hydrophobic components of the cell surface, as well as the properties of auto-agglutination and calcium dependence

However, this strain (as well as the parent No. 955S) contains a low molecular weight R-plasmid that controls its resistance to streptomycin. It is also resistant to kanamycin, beta-lactam antibiotics, erythromycin and lincomycin; sensitive to tetracycline, chloramphenicol, polymyxin.

The strain is obtained by breeding on a calcium-deficient environment from a population of the original calcium-dependent strain 9555, isolated in 1988 in P from a patient with yersiniosis, Strain 955S is typical for the properties of serovar 03; it is characterized by the presence of a plasmid 40-48 MD and some associated signs of virulence, auto-agglutination and calcium dependence.

Unlike the parent strain, strain 955L has a reduced potential pathogenicity. It does not show virulence in conjunctival infection of guinea pigs and enteral infection of mice. When infected with a tissue tissue HEp-2 (Xx xYc / ml), moderate adhesion and invasion of epithelial cells is noted, there is no intracellular reproduction.

The use of strain GISS Ms 189 is carried out as follows.

The pure culture of the strain is grown on solid nutrient medium (MPA, Hottinger) at 37 ° C for 18-24 hours. Using a 0.85% sterile solution of sodium chloride, the bacteria are washed off the agar and washed twice in the same solution by centrifuging (5000dh15min). Then the suspension is used for setting RA either directly (as a live culture) or after treatment with formalin (at a final concentration of 0.2 vol.%) For 20-24 hours at room temperature (as an inactivated culture). Prior to use, the suspension concentration is adjusted according to the optical turbidity standard to 10 cells / ml. The agglutination reaction with serum and the prepared antigen is set and taken into account in a conventional manner, as a rule, by mixing 0.5 ml of antigen with the same volume of serum in dilutions from 1:25 to 1: 1600. A positive response in a titer of 1: 200 and higher, as well as the dynamics of titers in paired sera, allow us to diagnose yersiniosis.

Example. Patient K., 12 years old. with a moderately severe intestinal infection, Y.enterocolitica serovar 03 was isolated from feces. At the end of the 3rd week of illness, serodiagnosis was taken. After separation from the clot, the serum is diluted with a 0.85% solution of sodium chloride from 1:25 to

1: 600 in a volume of 0.5 ml by two-fold dilutions. A culture of pieces was grown on Hottinger agar at 37 ° C for 24 hours. GISK No. 189. After the washout was prepared and bacteria were washed in physiological solution, the suspension was standardized according to the optical turbidity standard to 109 kg / ml. The finished 0.5 ml antigen is added to all dilutions of the patient’s serum, except

0 1:25, where added 0.5 ml of saline (control serum). In a separate tube mixed with 0.5 ml of suspension of antigen and 0.5 ml of saline (antigen control).

5 For the production of RA with a known antigen on Hottinger's agar at 24 ° C, a culture of pieces was grown for 36 hours. No. 134. After the washout was prepared, the culture was suspended in saline solution up to 109 cells / ml and 0.5 ml each.

0 was added to test tubes of a different range of the same dilutions of the patient's serum, and the corresponding control of the antigen was set.

An agglutination reaction with antigens from cultures was set up in a similar way:

5 Y.enterocolitica Ms 134, Y.enterocolitica of serovar 09 No. 201L, as well as E. coli and S. typhi grown on Hottinger agar at 37 ° C for 24 h. After shaking the tubes, incubated for 2 h at 37 ° C and 18 h at room temperature. The results are shown in the table.

It should be noted that the antibody titer in relation to the pre-strain of the strain is slightly higher than the titer to the reference strain Mg 134, although in both cases the antibodies are determined in diagnostic titers.

Antibodies are also detected by cross-reacting antigens: in the diagnostic titer (1: 200) - to plasmid-free

0 strain of Yersiniosis 09 pathogen, in low titers - to other enterobacteri m.

The appearance of antibodies to the pc. GISC No. 189 in high titers makes it possible to serologically confirm the diagnosis of Yersiniosis 03 in a large patient K.

Example 2. Patient B., 34 years old, with a mild course of bacteriologically confirmed yersiniosis (identi- fied hierosynyi serovar 03) at the beginning of the 3rd week

0 diseases taken blood for serological research. Twice separations of blood serum were prepared from 1:25 to 1: 1600 in a volume of 0.5 ml. A culture of pieces was grown on MPA at 37 ° C for 18 h.

5 NIS 1884 HISSTER. After the washout has been prepared and washed in a nat. the cell suspension solution is standardized to 109 cells / ml. Then formalin is added to it up to 0.2% (by volume); inactivated antigen was used one day after aging.

suspensions at room temperature. In a volume of 0.5 ml antigen was added to test tubes with serum dilutions; serum and antigen controls are set as usual. The test tubes were incubated for 2 hours at 37 ° C and 20 hours at 20 ° C. When taken into account, a diagnostic antibody titer of 1: 200 was detected. Parallel formulation of PA with a similar antigen from pcs. Mg 134 allowed detection of antibodies, but in the titer below diagnostic (1: 100), Therefore, when using antigen from pcs. GISS No. 189 confirmed the diagnosis of christiosis,

Thus, the strain GISK Mg 189, proposed for use as an antigen for the purpose of serodiagnosis of yersiniosis, has the following advantages as compared with the prototype.

When using pcs. GISC Ns 189 makes it possible to maintain a smooth form of Yersinia at 37 ° C, the absence of the masking effect of the H antigen and the antigens associated with the virulence plasmid increases the specificity of the resulting

ten

15

20

25

for serodiagnosis of PA, antibodies are detected in higher titers than with the prototype, which increases the frequency of serological confirmation of yersiniosis. Especially in doubtful cases, the Strain is isolated on the territory of the USSR and has typical biological characteristics for Yersinia Seravar 03 circulating in the country. The proposed strain is not susceptible to dissociation when cultured on nutrient media and does not require labor-intensive cloning to restore the S-shape. The pronounced activity of PCSIS No. 189 allows using the antigen prepared from it as an active drug for RA. Working with the proposed strain is safer for personnel.

Claims (1)

Claims of the invention Bacterial strain Yersinia enterocolitica GISC Mg 189 serovar 03 used as an antigen for serodiagnosis of yersiniosis The results of the accounting of the agglutination reaction of the serum of the patient K. with the Yersinia antigens of serovars 03, 09 and other enterobacteria Antigen, preincubation tera, ° C Reaction titer 1800 1: 400 1: 200 Less than 1: 100  In the control of serum flakes are absent, the liquid is clear; not subject to accounting Antigen control Uniform dregs Flakes on the bottom Uniform dregs