Monitoring of Diphtheriae Strains
Monitoring of Corynebacterium Diphtheriae Strains Circulating in Russia
Tech Area / Field
- MED-DIS/Disease Surveillance/Medicine
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
8 Project completed
Senior Project Manager
Evstyukhin K N
State Research Center for Applied Microbiology, Russia, Moscow reg., Obolensk
- Gabrichevsky Institute of Epidemiology and Microbiology, Russia, Moscow
- Centers for Disease Control and Prevention (CDC) / National Center for Infectious Diseases / Meningits and Special Patogens Branch, USA, GA, Atlanta
Project summaryThe aim of the project: to study molecular-biological and pathogenic properties of C. diphtheriae strains, circulating in Russia in the period of epidemic rise and recession of diphtheria during 1991-1998.
In the last years in Russia and other CIS countries epidemic situation with diphtheria became acute owing to socio and economic crisis, deterioration of ecological situation and conditions of life, migration of the population from diphtheria risky regions, decrease of the level of collective immunity. The morbidity rate has sharply increased, and in 1993 the infection has got the character of epidemic, during which serious omissions in diagnostics, preventive maintenance and treatment of this dangerous disease became clear. The experts note, that the level of diagnostics of diphtheria during the whole epidemic period remained low. The estimation of specific immunity of vaccinated persons and determination of the virulence of diphtheria causative agent in majority of cases is limited to definition of the titres of antitoxic immunity or the level of Corinebacterium diphtheriae strain toxigenicity.
In the system of epidemiological control of diphtheria infection in Russia the item on the supervision for circulating C. diphtheriae strains is specially pointed, since biological properties and possible variability of these strains can influence the development of epidemic and infection processes. Some aspects of the influence of biological characteristics of C. diphtheriae on the intensity of epidemic and infection processes are currently known. It is established, that at high morbidity rate, circulate mainly the strains characterized by certain biovar, high level of toxin formation, similar genetic structure. At the same time, for estimation of the possibility to use the data on biological properties of C. diphtheriae strains for forecasting the infection outbreaks and for the development of measures on the elimination of diphtheria causative agent, more profound researches on the biology of C. diphtheriae are necessary.
Within conducting of the project main attention will be paid to monitoring of diphtheria microbe, study of its molecular-biological properties, including virulent. Alongside with the researches of a wide spectrum of characteristics, having epidemiological significance and enabling one to make a reliable typing of the isolated strains, to find out sources and ways of infection, an increased attention will be focused on profound study of virulent and pathogenic properties of the dominant strains.
Technical Approach and methodology:
Cultural-morphological properties, biochemical activity with respect to glucose, starch, saccharose, cystine and urea as well as nitrate reduction of C. diphtheriae strains depoted in the collection will be investigated by using of liquid and solid culture media. For determination of toxigenic properties of the strains along with precipitation reaction on agar (Eleck test), PCR, reaction of indirect hemagglutination with erythrocytes sensibilized by antibodies to DT, solid-phase "sandwich" variant of ELISA with the use of monoclonal antibodies to the COOH-terminal site of DT B-fragment will be used. The LT of C. diphtheriae strains will be evaluated as average meanings of the reverse titres of the RNHA.
For intraspecies differentiation and typing of C. diphtheriae strains, besides pision into biovars and serotyping, methods of gene typing, blot-hybridization by using universal pRNA zond (ribotyping), multilocus enzyme electrophoresis (enzymetyping) will be applied as well.
For clearing the reasons of non-toxigenicity of C. diphtheriae strains, bearing DT gene, it is supposed to carry out the screening of non-toxigenic strains by PCR. To study PCR positive non-toxigenic strains by PCR associated analysis of conformational polymorphism (PCR-SSCP) as well as by direct sequencing with subsequent determination of their nucleotide sequences and comparison of the latter with analogous sequences of pW8 strains will be used.
In order to enrich methodical potentialities on profound and versatile study of the most epidemically significant C. diphtheriae strains of dominant ribo- and enzyme types, within the framework of the project it is planned to perform experiments on revealing and researching of the potential virulence factors of diphtheria microbe, preparation of diagnosticums for their control. At present DT of C. diphtheriae is investigated most profoundly. The other virulence factors, participating in survival and multiplication of the bacteria, in patho- and immunogenesis of diphtheria infection are not practically investigated at monoantigen level. For realization of this stage of researches it is supposed to isolate and characterize highly purified major antigens, including surface secreted and membrane proteins as well as proteins possessing of enzymatic activity, to evaluate their protectivity on laboratory animals. Monoreceptor serum and suspension (erythrocytic and latex) diagnosticums for control and investigation of antigens and enzymes will be prepared.
For isolation and purification of antigens, a principle of step- extraction of surface secreted and membrane proteins followed by reversed-phase HPLC will be used. Monoreceptor diagnosticums will be prepared by immunization of rabbits with purified proteins or their mixes with subsequent removal of moisture antibodies and isolation of gamma-globulin fraction. Suspension diagnosticums will be prepared by sensibilization of latex particles and formaline treated erythrocytes by gamma-globulines and purified monoantigens.
Estimation of the functional importance of antigens as potential virulence factors will involve studies of their adhesive and antiphagocytic properties, enzymatic activity and toxicity. Attempts to obtain mutants defective in the synthesis of separate antigens and enzymes with subsequent study of their virulence properties on laboratory animals will be made.
At the final stage of the researches by scaling data on monitoring C. diphtheriae strains and diphtheria infection comparative analyses of molecular and biological properties of pathogen regarding severity of caused disease, induction and intensity of immunity in sick patients will be conducted.
As the result of the project fulfilling it is expected to enrich C. diphtheriae strain collection; to characterize genetic structure of C. diphtheriae strains, circulating in various periods of diphtheria epidemic; to study the structure of DT (tox) gene and its regulatory element (dtxR) in nontoxigenic strains, bearing tox gene; to develop recommendations for perfection of methods of diagnosticis, intraspecies typing, prophylaxy and treatment of the sick and bacterium carriers in order to eliminate diphtheria causative agent from human population; to reveal and characterize bacterial factors of virulence and immunogenicity of C. diphtheriae; to evaluate the possibility to use the molecular-biological characteristics of the microbe for forecasting the intensity of infection and epidemic processes; to formulate conception of molecular monitoring of C. diphtheriae and togive based recomendation on perfection of means and methods of diphtheria diagnostics.
The planned researches meet the objectives of ISTC and will promote reorientation of the experts, early working on a defense program, on the decision of fundamental and applied problems of medicine, as well as integration of them with international scientific community.
Potential role of collaborators:
— consulting and methodical assistance;
— joint researches on ribo- and enzyme typing of epidemic strains of C. diphtheriae;
— cooperative study of the structure of tox and dtxR genes of nontoxigenic strains of C. diphtheriae.