The project aims to improve an epidemiological surveillance of anthrax, brucellosis and campylobacter infections spread among farm animals and the human population of the Issyk-Kul region of Kyrgyzstan. This will be done on the basis of serological, molecular and GIS technologies.
Dangerous infections such as anthrax, brucellosis, plague constitute acute public health problems for the Kyrgyz Republic (KR). The epidemiological situation of anthrax in the Republic in the last 30 years is gradually deteriorating due to the degradation of its economic state. 1,241 stationary foci of anthrax have been registered in Kyrgyzstan and 114 of them are located in the Issyk-Kul region. The absence of correct epidemiological control led to an annual increase in the number of reported human anthrax outbreaks. Thus, in 2013 16 cases of human anthrax have been reported in Kyrgyzstan, although none were reported in the Issyk-Kul region. Today the number of found foci is very low: from 114 known only 40 are registered, fenced and mapped, the rest over 50% still remains not found. In the same time the overall climate change creates favorable conditions for the preservation and development of B. anthracis spores and other pathogenic organisms. The climatic features of the Issyk-Kul region, including mudflows, floods, landslides, coastal erosion, storm flooding, hurricane, hail, landslides, drought seasons, earthquakes, can brings spores to the surface of soil. This can lead to outbreaks of anthrax, even in areas that for a long time are considered relatively safe. In this situation, capacities for a quick and accurate identification of anthrax are urgently needed. The method for determining the contamination level of anthrax spores in the soil - Ground anthrax bacillus refined isolation (GABRI) developed by our Italian collaborator will be applied. This will represent for Republic a fundamentally new development and will significantly improve the control of potentially dangerous territory.
Beside anthrax the project involves a simultaneous epizootic and epidemiological study of brucellosis of farm animals and human population of three livestock districts of the Issyk-Kul region, which have not undergone such controls in the past 30 years. In total 35 settlements with the highest number of cases of brucellosis registered in the last five years will be covered by molecular and serological diagnostics. The basic screening coverage of cattle, small cattle and horses will be performed on the area of Kyrgyz traditional summer pastures - in high mountain syrts Ak-Suu, Jety-Oguz and valleys Kok-Zhaik, Karkyra.
Another very important problem in Kyrgyzstan are the acute enteric infections (AEI) of unknown pathogenesis that have increased dramatically within the last 25 years and constitute 39% of the total number of all infections. Among them the major infection could be campylobacteriosis with Campylobacter jejuni as a main causative pathogen (Gillespie et al, 2002). The local bacteriological laboratories do not have an adequate equipment to perform the identification of pathogens. In these cases the treatment of patients (mostly children of 0-2 years age) is not correct and may finish with death - this situation results in more as 200 lethal cases per year. Again, for the correct treatment the resistance of isolated strain to different antibiotics should be analyzed. Such methods are currently not available because of the financial deficit of most state laboratories in KR.
The project will allow the accurate identification of the causative agents of anthrax, brucellosis and campylobacteriosis in the Issyk-Kul region of Kyrgyzstan. The planned work will contribute to the control of the spread of infection and help establish biosafety and biosecurity protocols in the region between two neighboring countries - Kyrgyzstan and Kazakhstan that have longstanding trade ties in the dairy and meat food industry.
The researchers who will participate in the project have experience in the study of the epidemiology of brucellosis and epizootics in high mountain syrt area (summer pasture), identification of the causative agent by traditional methods (bacteriological, serological) and thematic mapping of the territory. This is confirmed through their participation in international projects, including projects funded by ISTC (#KR-1784 "Investigation of epidemiology and epizootiology of plague for the purpose of biosafety of the population of the Issyk-Kul region of Kyrgyzstan", #KR-2111 “Molecular - genetic monitoring and passportization of transboundary Sari-Dzhas focus of plague with GIS-technologies in Kyrgyzstan and Kazakhstan“). These projects are undertaken jointly with the Karakol department of the quarantine and especially dangerous infections. The IDCVDE personnel participated to the 61th International Conference "Brucellosis 2008", organized by the Royal Holloway College and the University of London and are co-founders of the Association of Biosafety in Central Asia and the Caucasus (BACAC). The implementation of the proposed project will promote international technical and scientific cooperation in the field of epidemiology and epizootiology of brucellosis, anthrax and campylobacter associated diseases and the creation of long-term prospects for the control of infection in the framework of the International Association of Scientific and BACAC.
The first international collaborator will be Prof. Heinrich Neubauer, Head of the Friedrich-Loeffler-Institut from Germany. The Institute of Bacterial Infections and Zoonoses hosts the German National Reference Laboratory for Brucellosis as well as an OIE Reference Laboratory for Brucellosis. The labs are well equipped and staff is experienced in all state of the art diagnostic methods to detect brucellosis and identify and type Brucella spp. The scientists have many experiences in co-operation with other institutions all over the world, have performed training courses in the frame of twinning projects and are responsible for examination of samples from countries which are members of the OIE. Scientific work results in many publications concerning brucellosis. The staff will perform laboratory training for two weeks for project participants learning the techniques of identifying and typing of Brucella spp.
The second international collaborator – Dr. Gilles Vergnaud formerly headed a research Group of Bioinformatics, genomes and evolution in the Institute of Genetics and Microbiology of University Paris-Sud (France). He currently is Dean of Education and Research of ENSTA ParisTech which is a college of engineering within the newly created University of Paris Saclay. He is a very well known expert in the area of genotyping of many types of bacteria and will fill in a course for data management of genotyping data with a focus on Brucella for one week. The third international collaborator – Dr. Antonio Fasanella is Responsible of the Anthrax Reference Center of Italy. He was a collaborator of the previous ISTC project #KR-1632 “Assessment of Spatial Mechanisms of Pollution of the Territory of Kyrgyzstan by Anthrax Agent”. His group will organize a course on the new developed high effective GABRI method of isolation of anthrax from soil samples and their identification for 1 week.
The soil probes from known anthrax foci for identification will be taken in the Ak-Suu, Jety-Oguz and Tup districts. The probes will be prepared for GABRI analysis as described by Fasanella et al., 2013. The identification of DNA will be performed by Anthrax-specific PCR (Fasanella et al., 2001). MLVA analysis and Canonical SNP will be done on the isolated strains of Bacillus anthracis to describe a map of the genotypes of B. anthracis circulating in the Kyrgyz Republic.
The molecular and serological analysis of blood for the presence of Brucella will be conducted in the laboratories of the Issyk-Kul district center of veterinary diagnostics and expertise. The mobile group for collection of animal blood will be made from three persons: one bacteriologist and two technicians. The work time duration of the group is 20 days in the summer season (roughly from May 25 to June 15). For the maximum effective collection of clinical material three groups will be constituted each year in Ak-Suu, Jety-Oguz and Tup areas. During autumn – spring the blood from animals will be collected regularly directly from the field with the help of local veterinarians and delivered weekly to the laboratory for analysis. Approximately 35 human settlements and the corresponding 36,000 head of cattle, horses and sheep will be covered. In general, it is planned to carry out the diagnostics of 70,000 – 80,000 serums of blood in three years.
The diagnostics of brucellosis in the human population of the three selected areas will be carried out by molecular and serological methods followed by the clinical indicators of hospitalized patients and the symptoms of brucellosis by baseline medical examination. The patients coming from local branches of the Family Medicine Centers will also be investigated. Overall, 4000 human blood samples will be examined per year.
The serological method of brucellosis diagnostic will follow the Guidelines for the diagnostics of brucellosis in animals developed by the Ministry of Agriculture, Water Resources and Processing Industry of the Kyrgyz Republic, Bishkek, №01-15-1337/1 from 14.10.2009. The basis of this method is a determination of antibody binding to an antigen in the reaction of Wright - Heddlson (by human), agglutination test in tubes (RA), reaction of complement fixation (RCF), reaction of long complement fixation (RDSK), plate agglutination test with Rose Bengal antigen (Rose Bengal sample) for animals. In the frame of the project it is planned to use commercially available ELISA kits (e.g. from IDEXX or IDVet, etc.). Specific kits to examine milk (milk bulk) or serum are available. DNA for PCR diagnosis will be extracted from sample material using standard kits (e.g. Qiagen «QIAamp ® DNA Mini Kit»). PCR reaction will be carried out by a real-time PCR method published by of Probert et al. (2004). The method includes three multiplexed PCR to examine a sample for a Brucella genus-specific DNA sequence as well as specific sequences to determine whether Brucella melitensis or Brucella abortus is present. In case the sample would be only positive in the genus specific test the system could be modified to find B. suis specific DNA also. All of these analyzes will be carried out in the laboratories of the Issyk-Kul zonal center and veterinary diagnostics expertise and Karakol center of disease prevention and the state sanitary and epidemiological surveillance.
The Geographic Information System (GIS) ArcGIS 10.1 and Arcmap will be used to create the electronic maps and database of anthrax, brucellosis and campylobacter in the Issyk-Kul region. Software ArcGIS 10.1, and Arcmap will be used as Geographic Information System (GIS). The areas with highest risk of anthrax, brucellosis and campylobacter infections in the Issyk-Kul region will be mapped using the Arc GIS 10 software. The database will contain coordinates of epizootics areas, epidemiological survey data, data of epidemic complications from 1998 to 2013. The system is developed based on GIS - platform ArcView. The electronic maps of Kyrgyzstan M 1:100,000 will be used as topographic base will be used electronic maps of Kyrgyzstan M 1:100,000.
