The project involves entire molecular - genetic monitoring of the Sari-Dzhas transboundary natural plague focus located in the Kyrgyzstan and Kazakhstan and its electronically passportization using GIS – technologies. An acute epizootic of plague which was transmitted to human and followed a death of patient was registered in the Sari-Dzhas (Enylchek-Kaindy area, Kyrgyzstan) in 2012-2013 for the first time since 1982.
In framework of the project the remote and contiguous territories of transboundary Sari-Dzhas focus of plague will be jointly studied by Kyrgyz anti-plague service and Kazakh experts by molecular - genetic and GIS- methods. For the first time in Kyrgyzstan the actual isolates of plague will be analyzed by MLVA-25 method and compared with that from Kazakhstan, Russia, and China.
First in Kyrgyzstan we will conduct the molecular screening not only in animals - the carrier of the pathogen, but also in their ectoparasites. This will allow the complex monitoring of entire natural cycle of plague infection: marmot - ectoparasite - marmot, identifying of potentially dangerous sectors, improving of diagnostic and better control of plague biorisk in both countries. Creating of an electronic data base system (database on epizootiology and epidemiology of plague), visualization of this information on a map of the Issyk-Kul (Kyrgyzstan) and Almaty (Kazakhstan) regions will help to evaluate the policy for biosafety of population in the border region between 3 neighboring states: Kyrgyzstan, Kazakhstan and China. Similar work in Kyrgyzstan has not yet been carried out.
The scientists that will take part in this project are well experienced in the investigation of plague epizootics in the mountain area, including identification of pathogens by traditional (bacteriological, serological, biological), genetic methods (MLVA-25), and GIS-passportization. This is evidenced through participation in several international projects, including projects financed by ISTC #KR-1784 “Investigation of plague epidemiology and epizootiology for the purpose of biosafety of population in Issyk-Kul region of Kyrgyzstan”; # KR-330.3 “Studying Water Balance and Hydrodynamics of Lake Issyk-Kul with a Help of Isotope Methods”; in the program of Biological Threat Reduction of USA Department of Defense № KZ-28 "Genetic features of particularly dangerous zoonotic pathogens (Yersinia pestis, Francisella tularensis, Bacillus anthracis) of Kazakhstan»; SBI-TAR-6 «Immunodiagnostic of antiplague vaccination of human in Kazakhstan» and interregional workshops on “Biosafety risk assessment” organized by the “Biosafety Association for Central Asia and the Caucasus”. The activities of proposed project will help to promote international scientific and technical collaborations in the field of plague epidemiology and create long-term prospects for control of the disease spread within the framework of International Scientific Association.
The foreign collaborators, Dr. Vladimir L. Motin of Medical Branch of the University of Texas, and Dr. Michael Kosoy of the CDC/OID/NCEZID, will evaluate the activities of the project team as experts in the following areas: analysis of isolated strains by MLVA-25 method of genotyping, phenotype characterization, multifactor analysis of the obtained results and use of GIS-technologies to create passports and databases, as well as in the conduct of molecular monitoring of animals and ectoparasites. Foreign collaborators will assist by selection of equipment and chemicals, research development, intellectual property and information support of the project, reviewing and editing of scientific publications based on the results of the project, holding discussions on the key issues of the project.
The monitoring of plague epizootic at the natural source area will be conducted with a help of Kazakh experts during an epidemiological expeditions and involve the hunting of marmots and other rodents as a plague reservoir and their analysis on pathogen presence. Biological, bacteriological and serological analyses will be carried out in the field laboratories during expedition. The epidemiological mobile group will consist of 16 people made up of: two baсteriologist, zoologist, parasitologist, 2 technical assistants, 6 catchers/hunters of marmot and rodents, 1 groom, 2 drivers, 1 cook. The work duration of the group will be 40 days in summer (approximately from May 25 until July 5). The Kyrgyz epidemiological group in the first year will work in the Koyluu place because here in 2013 was registered a human plague; in the second year – in the Kok-Dzhar area, where the last epizootic was shown in 1974; and in third year – in the Enylchek-Kaindy area with 26 cultures isolated in 1977 and 5 in 2012. The whole area of investigation for three years will be 2500-3000 sq. km of mountain valleys, gorges and rivers. The Kazakh expedition of the same composition will work at the Big and Small Kokpak sub regions with total area of 1750 sq.km. This investigation allows entire monitoring of whole area of transboundary Sari-Dzhas plague focus.
Determination of the presence of the plague pathogen will be based on serological, biological, and bacteriological testing according to the methods for diagnosis, treatment and prophylactics of plague, as described in the All-union Anti-plague Research Institute Manual “Microbe”, Saratov, Russia, 1974, 1977. For the biological method, the sterile white mouse bioassay will be used. The bacteriological method will be based on the susceptibility of the culture isolates to the plague bacteriophage (pure and diluted 1:10, 1:100, and 1:1000) and to the bacteriophage L-413 “C”. In addition, the diagnosis of plague will be conducted based on the typical morphology of the cells in smears from animal samples, typical morphology of the growing cell colonies, peculiarities of the cell growth in broth, pathological and anatomical changes of investigated animals, presence of pathogens of certain form and color in the smears from the organs of investigated animals, and isolation of the plague bacteria from culture. For evaluation of the phenotype of isolated strains will be used testing for pigmentation, fibrinolysis, fermentation of glycerol, rhamnose and arabinose, production of capsule.
The Multi-locus VNTR analysis (MLVA) with 25 loci will be performed in the state laboratory of ISU on DNA extracted from animals and ectoparasites without exchange of strain, using a standardized protocol (Motin at al., 2002; Pourcel et al., 2004; Li et al., 2009). The genotyping will be performed on a standard vaccine strain EV76 from Kazakh Scientific Center for Quarantine and Dangerous Diseases, Almaty, Kazakhstan. Following MLVA clustering will allow the analysis of the genetic diversity and geographic origin of the isolates and comparing of them with known isolates of Kazakhstan, Russia and China.
The long term storage of the plague specimens in the case of positive testing of field samples will be carried out at the main repository of the Republican Centre for Quarantine and Especially Dangerous Infections in Bishkek in accordance with Instructions of the Ministry of Health Service of Kyrgyzstan.
To create the electronic maps of Sari-Dzhas plague focus with coordinates of epizootics areas, epidemiological survey data, data of epidemic from 1954 to 2013 will be used geographic information system (GIS) software ArcGIS 9.1, Arcmap with. The system is developed based on GIS - platform ArcView. As a topographic base will be used electronic maps of Kyrgyzstan and Kazakhstan M 1:100,000.
