RU2560581C2 - AGENT FOR PRODUCING PREPARATIONS FOR DIAGNOSTICS OF RICKETTSIOSES CAUSED BY RICKETTSIA SIBIRICA subsp. SIBIRICA BJ-90 - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and concerns strain Rickettsia sibirica subsp. sibirica. Described use of strain R. sibirica subsp. sibirica "Primorye-32/84" of genotype BJ-90, deposited in the All-Russian museum of rickettsial cultures of FGBU NIIEM named after N.F. Gamalei with the accession number of 160, for the development of the preparations for diagnostics of rickettsioses caused by Rickettsia sibirica subsp. sibirica BJ-90. The strain was isolated in the RF from imago ixodic ticks D. silvarum, collected in Primorsky Krai in 1984. The strain is a pathogen for guinea pigs, successfully cultivated on cell culture Vero E6.

EFFECT: solution may be used for identification of tick spotted fever rickettsias on the basis of genotypic and phenotypic characters and for producing diagnostic preparations.

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Description

The invention relates to the field of biotechnology and is an original strain of rickettsia group of tick-borne spotted fever "Primorye-32/84". The strain "Primorye-32/84" is a representative of the genotype BJ-90 of the species Rickettsia sibirica subsp. sibirica, first isolated on the territory of the Russian Federation. Based on the study of phenotypic and genotypic characters, the strain is assigned to the Rickettsiales order of the Rickettsiaceae family, the genus Rickettsia, and the species Rickettsia sibirica subsp. sibirica genotype BJ-90.

R.sibirica includes two subspecies: R.sibirica subsp. sibirica, the causative agent of Siberian tick-borne typhus (SKT) and R. sibirica subsp.mongolotimonae, is the etiological agent of "associated with lymphangitis rickettsiosis" ("lymphangitis-associated rickettsiosis" or LAR). The first of these subspecies was first isolated in Russia and later discovered in northern China (Yu X., Y. Jin, M. Fan, G. Xu, Q. Liu, and D. Raoult. 1993. Genotypic and antigenic identification of two new strains of spotted fever group rickettsiae isolated from China. J. Clin. Microbiol. 31: 83-88). R. sibirica subsp. mongolotimonae was first isolated in Inner Mongolia and then identified in southern Europe and Africa (Fournier R.E., F. Gouriet, P. Brouqui, F. Lucht and D. Raoult. 2005. Lymphangitis-associated rickettsiosis, a new rickettsiosis caused by Rickettsia sibirica mongolotimonae: seven new cases and review of the literature. Clin. Infect. Dis. 40: 1435-1444, 12). In 1991, the rickettsial strain with unknown pathogenicity, Rickettsia sp. strain BJ-90 isolated from Dermacentor sinicus ticks collected around Beijing, China in 1990 (Yu XJ, Fan MY, Bi DZ 1991. Isolation and primary identification of spotted fever group rickettsiae BJ-90 strain. Chin. J. Microbiol Immunol. 1:31). Phylogenetically Rickettsia sp. strain BJ-90 is close to Rickettsia sibirica subsp.sibirica and R.sibirica subsp. mongolotimonae, forming a single cluster with them, however, is more closely associated with Rickettsia sibirica subsp.sibirica and is recognized as a genetic variant of this rickettsia species (Zhang JZ, Fan MY, Yu XJ, Raoult D. Phylogenetic analysis of the Chinese Rickettia isolate BJ-90. Emerg Infect. Dis. 2000, 6: 432-433). In China, strains of the BJ-90 genotype were isolated from only one tick species, D.sinicus, and in the same area for several years (Zhang JZ, Jin JL, Fu H.P., Raoult D. and Fournier, PE Genetic differentiation of Chinese isolates of Rickettsia sibirica by partial ompA gene sequencing and multispacer typing. 2006. J. Clin. Microbiol. 40: 2465-2467).

The incidence of rickettsiosis caused by the BJ-90 genotype of the species Rickettsia sibirica subsp. sibirica, is currently not registered in Russia, since diagnostic products for verification of this rickettsiosis in the Russian Federation are not available. The diagnosis of rickettsiosis associated with the BJ-90 genotype of the species Rickettsia sibirica subsp. sibirica, can only be delivered by laboratory examination using test systems based on production strains of the BJ-90 genotype of the species Rickettsia sibirica subsp. sibirica.

Rickettsia strains of genotype BJ-90 of the species Rickettsia sibirica subsp. sibirica, available in the collection of the Omsk Research Institute of Natural Focal Infections, is the first strains of this genotype of rickettsia isolated from Dermacentor silvarum ticks outside of China. The strain of the BJ-90 genotype of the species Rickettsia sibirica subsp. sibirica "Primorye-32/84" was isolated from adults of ixodid ticks D.silvarum collected in the Dalnerechensky district of Primorsky Krai in 1984, i.e. 6 years earlier than the isolation of the first Chinese strains of this rickettsia.

The strain "Primorye-32/84" is cultivated in a culture of Vero cells, chicken embryos, guinea pigs.

According to the results of studying and comparing the nucleotide sequences of gltA and ompA gene fragments, it has 100% homology with a typical strain of the BJ-90 genotype of the species Rickettsia sibirica subsp. sibirica.

The strain of the BJ-90 genotype of the species Rickettsia sibirica subsp. sibirica "Primorye-32/84" is stored in the All-Russian Museum of Rickettsial Cultures FSBI NIIEM them. N.F. Gamalei under inventory number 160 dated March 4, 2013. The objective of the invention is the original strain of the genotype BJ-90 of the species Rickettsia sibirica subsp.sibirica "Primorye-32/84", which can be used for the development and manufacture of diagnostic preparations (sets of reagents).

The technical result is the use of a strain of the genotype BJ-90 of the species Rickettsia sibirica subsp. sibirica "Primorye-32/84", as a production strain in the development and manufacture of preparations for the diagnosis of rickettsiosis caused by the BJ-90 genotype of the species Rickettsia sibirica subsp. sibirica. The inventive strain has the ability to actively reproduce in various biological systems (guinea pigs, developing chicken embryos, Vero cell culture) and accumulate production biomass during cultivation, which allows it to be used for serial (commercial) production of diagnostic preparations (reagent kits). Using a strain of the BJ-90 genotype of the species Rickettsia sibirica subsp. sibirica "Primorye-32/84", as a producer of diagnostic products, allows optimizing laboratory diagnosis of rickettsioses and monitoring of combined natural foci of rickettsioses of the group of tick-borne spotted fever (KPL) in Russia.

An isolated strain of the BJ-90 genotype of the species Rickettsia sibirica subsp. sibirica "Primorye-32/84" is characterized by the following features:

Morphological signs. Typical for rickettsia, but rod-shaped forms are more common than others (type b according to the classification of P.F. Zdrodovsky). Gram-negative.

Cultural properties. It is successfully passaged in Vero-E6 cell culture, with a maximum accumulation of rickettsia up to 25-30 or more copies in sight. When cultivated on 5-6 day old chicken embryos, starting from the second passage it gives an accumulation of 50 or more rickettsia in the field of view.

Passage characteristic. The strain was isolated in 1984 in a biological test on male guinea pigs and, after eight passages in guinea pigs, was transferred to chicken embryos. The culture of the strain of the genotype BJ-90 of the species Rickettsia sibirica subsp. sibirica "Primorye-32/84" is lyophilized dried on the 5th passage on chicken embryos.

Antigenic properties. Antigenic determinants of the BJ-90 genotype of the species Rickettsia sibirica subsp. sibirica are detected by the method of fluorescent antibodies with diagnostic immunoglobulins to detect rickettsia of the CPL group.

Immunogenic properties. In the complement binding reaction (CSC) with the R.sibirica antigen and guinea pig sera infected with the BJ-90 strain of the species Rickettsia sibirica subsp. sibirica "Primorye-32/84, seroconversion in titers of 1: 40-1: 80 is observed. When ELISA examines the blood sera of febrile patients who had a history of sucking ticks with the BJ-90 genotype antigen of the species Rickettsia sibirica subsp. sibirica, prepared from strain "Primorye-32/84", antibodies of the IgM class to this type of rickettsia were detected in 16.0 ± 3.2%, antibodies of the IgG class in 9.9 ± 2.6%. There is a lack of cross-reactions in the study of blood serum of patients tick-borne rickettsiosis for the presence of antibodies of the IgM and IgG classes in ELISA with antigens of R. sibirica subsp. sibirica and R. sibirica subsp. sibirica genotype BJ-90, which allows to evaluate the strain as a candidate for the development of diagnostic products.

Virulent properties. With intraperitoneal infection of guinea pigs, the strain causes a 2-3-day febrile reaction with severe periorchitis. At autopsy in animals, a pathological anatomical picture characteristic of tick-borne rickettsiosis is observed.

Genotypic characteristics. The nucleotide sequence of the synthase citrate synthase (gltA) gene was determined with a length of 1234 base pairs of the strain Primorye 32/84, which had 100% homology with the sequence of the citrate synthase gene of the type strain R.sibirica subsp. sibirica genotype BJ-90 deposited in GenBank under the number AF178035:

The nucleotide sequence of the rickettsia outer membrane protein (OmpA) gene length of 590 base pairs has 100% homology with GenBank member number AF179365 Rickettsia sibirica subsp. sibirica genotype BJ-90. OmpA (ompA) gene:

Based on morphological, antigenic, and genetic characters, an isolated strain is assigned to the genus Rickettsia, the species Rickettsia sibirica subsp. sibirica genotype BJ-90.

The invention is illustrated by the following examples.

Example 1. Identification based on genotypic traits. DNA extraction.

Rickettsial DNA was extracted from a cell culture sample using the QIAamp Tissue Kit (QIAGEN, Hilden, Germany) and proteinase K at a concentration of 2.0 mg / ml (Boehringer Mannheim, Mannheim, Germany) according to the QIAamp Tissue Kit protocol. Polymerase chain reaction (PCR) and sequencing reaction.

The polymerase chain reaction (PCR) and the sequencing reaction are performed using Rickettsiae-specific primers using two pairs of primers amplifying the gltA gene encoding citrate synthetase: CS1d (5-ATGACTAATGGCAATAATAA-3) and CS535r (5-GAATATTTATA ), CS409d (5-CCTATGGCTATTATGCTTGC-3) and RP1258n (5-ATTGCAAAAAGTACAGTGAACA-3), as well as primers 190.70 (ATG GCGAATATTTCTCCAAAA), 190.180 (GCAGCGATAATGGCTGTA protein), GTCGATGTGTAGTA code . et al., 1997; Fournier PE. et al., 1998).

PCR was performed on a Peltier thermal cycler model PTC-200 (Mj Research, Inc, Watertown, MA). The reaction mixture for each sample contains 10 pmol of each primer, 0.5 polymerase units (Elongase, GibcoBRL), 20 mM of each deoxynucleoside triphosphate, 1.8 mM MgCl ₂ and 7.5 μl of DNA from each sample, the final volume is 25 μl. Amplification is carried out according to the following program: 3 min of initial denaturation at 94 ° C, in the next 44 cycles 30 s of denaturation at 94 ° C, 30 s of primer annealing at 55 ° C, 90 s of elongation at 68 ° C and 7 min final elongation at temperature 68 ° С. Each PCR setup includes a negative control (distilled water) and a positive control (R.montanensis DNA).

Purification of the PCR product is carried out using the QIAquick PCR Purification Kit (Germany) in accordance with the protocol.

A sequencing reaction with PCR products was performed using the d-Rhodamine Terminator Cycle Sequencing Ready Reaction kit (Applied Biosistems, Warrington, UK). Sequencing is performed on an ABI 3100 PRISM (Applied Biosystems) in an automatic sequencer. Analysis of the obtained nucleotide sequences to compare the degree of homology with the sequences of the GenBank data bank is carried out using the BLAST program in direct access mode.

Example 2. Identification based on phenotypic (cultural and antigenic) properties. The cultivation of rickettsia.

Rickettsia is cultured on a Vero E6 cell culture. The culture was grown according to the standard method of Solovyov, Bektemirov 1972, as the growth medium used MEM Needle with a double set of amino acids with the addition of 5% fetal serum. The optimal seeding dose of 100-150 thousand cells per 1 ml. The next day after the formation of a monolayer, the cell culture is used for infection.

Infection of cell cultures.

The cell culture in the mattress is infected with a 50% suspension obtained from the dried strain culture at a dose of 1 ml per mattress. In all mattresses with infected cells, support medium (MEM needle with the addition of 1% fetal serum) is added in a volume of 9 ml per mattress (Raoult D., 2000). Mattresses with infected cells are cultured in a thermostat at a temperature of 37 ° C for 10-14 days. The maximum accumulation is observed from the second 2 passage and is 25-30 or more copies in each field of view, with the predominant localization of the microorganism in the cytoplasm of the cells. Infection and sterility of cell culture is determined in smears, staining them according to Romanovsky-Giemsa.

The obtained samples are examined by the method of fluorescent antibodies according to the standard method, diagnostic immunoglobulins are used to detect rickettsia of the CPL group, luminescent dry (FSUE NPO Mikrogen, Perm). The study of morphological and tinctorial properties is carried out according to the method of Zdrodovsky and Golinevich (1972), to confirm belonging to rickettsia, PCR with rhodospecific primers is used.

Example 3. Preparation of Fully Soluble Antigens

To prepare the antigen, the infected cell cultures are centrifuged at 6,000 rpm for 20 minutes, then the supernatant is removed, and smears are made from the resulting suspension, which are stained according to Romanovsky-Giemsa.

After microscopic control, physiological saline and 0.5% phenol are added to the suspension of infected cells. After shaking in a shaker, tubes with infected cell suspension are placed in a refrigerator for 3-4 days to precipitate foreign protein impurities.

After this, the cell suspension is subjected to repeated ether treatment according to Craigie (1945). During the first ether treatment, 1.5-2 volumes of anesthetic ether are added to the material and, after shaking, they are left at room temperature until the next day in a dark place. Then the aqueous phase is separated and treated with ether a second time (equal volume of ether in relation to the aqueous phase), shaken and left for 2 hours, then the aqueous phase is separated again and treated with ether for the third time (1/2 volume of ether relative to the volume of the aqueous phase) in the same way.

After the third treatment, a secondary ether treatment is done to form the middle tissue layer. After completion of the ether treatment, the ether is distilled off under a small vacuum. Test tubes with antigen are left in the refrigerator for one day to evaporate the ether. The resulting series of antigens are tested in the complement binding reaction of CSCs for anticomplementarity, specificity, sensitivity and a titer is established. Also, antigens are standardized by the level of amine nitrogen by formol titration (%) and protein using a biuret reaction (%). After the test, antigens are poured into 1.0 ml ampoules and freeze-dried. The dried antigen is stored at a temperature of -20 ° C.

Preparation of corpuscular antigen.

After cultivation, the culture of infected cells is frozen at -20 ° C, and then thawed to destroy the cells and maximize the yield of rickettsia from them. The material is centrifuged for 10 minutes at 3000 rpm. The supernatant is used to apply particle antigen to glass and conduct an indirect immunofluorescence reaction to study the sera of tick-borne infections.

Thus, the strain has 100% homology with a typical strain of the BJ-90 genotype of the species Rickettsia sibirica subsp. sibirica, based on a comparison of the nucleotide sequences of the gltA gene and ompA gene, actively propagates in Vero cell culture and in developing chicken embryos, which allows the accumulation of production biomass during cultivation. With this in mind, it can be used as a production strain in the development and manufacture of drugs for the diagnosis of rickettsiosis caused by the BJ-90 genotype of the species Rickettsia sibirica subsp. sibirica.

Claims (1)

The use of the strain Rickettsia sibirica subsp. sibirica "Primorye-32/84" of the genotype BJ-90, deposited in the All-Russian Museum of Rickettsial Cultures FSBI NIIEM them. N.F. Gamalei at number 160, for the development of drugs for the diagnosis of rickettsiosis caused by Rickettsia sibirica subsp. sibirica BJ-90.