Modern Diagnostic Technologies in the Study of the Field Material Collected from the Natural Plague Foci of the Kyrgyz Republic in 2023

The aim of the work was to analyze the results of investigating the field material obtained from natural plague foci of the Kyrgyz Republic (KR) in 2023, using modern diagnostic technologies.

Materials and methods. 1435 biological samples from the Tien Shan, Alai and Talas high-mountain foci of the Kyrgyz Republic were studied using conventional methods of laboratory diagnosis of plague: microbiological, immunological, biological; as well as modern molecular-genetic methods. Testing of the obtained samples for the presence of plague pathogen DNA was carried out using RT-PCR; and the presence of antibodies to the plague microbe was detected by enzyme-linked immunosorbent assay (ELISA). Molecular identification of Yersinia pestis strains according to their appurtenance to subspecies, biovars, and phylogenetic lineages was performed by RT-PCR using the method of identifying single-nucleotide substitutions based on the analysis of melting curves of products.

Results and discussion. An approach to the molecular identification of Y. pestis strains from plague foci of the KR has been developed and validated through identifying singlenucleotide substitutions using the analysis of product melting curves (HMR-analysis) with a set of designed primers. It has been established that Y. pestis strains isolated in the Sarydzhaz autonomous focus of the Tien Shan high-mountain focus belong to the biovar antiqua of the main subspecies, phylogenetic branch 0.ANT5. The phylogenetic relation of Y. pestis strains isolated in 2023 was studied based on genome-wide SNP analysis. Areas of epizootic activity in Eastern Alai have been identified. The data obtained indicate the sustained activation of plague foci in the KR. Areas of the territory of the KR that are promising for paleogenomic research are also discussed.

1. Popova A.Yu., Kutyrev V.V., editors. [Atlas of Natural Plague Foci of Russia and Foreign Countries]. Kaliningrad: RA Poligrafych; 2022. 348 p.

2. Popova A.Yu., Kutyrev V.V., editors. [Cadastre of Epidemic and Epizootic Manifestations of Plague in the Territory of the Russian Federation and Former Soviet Union (1876–2016)]. Saratov: “Amirit” LLC; 2016. 248 p.

3. Eroshenko G.A., Nosov N.Y., Krasnov Y.M., Oglodin Y.G., Kukleva L.M., Guseva N.P., Kuznetsov A.A., Abdikarimov S.T., Dzhaparova A.K., Kutyrev V.V. Yersinia pestis strains of ancient phylogenetic branch 0.ANT are widely spread in the high-mountain plague foci of Kyrgyzstan. PLoS One. 2017; 12(10):e0187230. DOI: 10.1371/journal.pone.0187230.

4. Eroshenko G.A., Kukleva L.M., Kutyrev V.V. [Historical and modern classifications of the plague agent]. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2022; (4):14–22. DOI: 10.21055/0370-1069-2022-4-14-27.

5. Abdikarimov S.T., Ibragimov E.Sh., Egembergenov Ch.E. [Current epizootic condition of natural plague foci in Kyrgyz Republic and measures aimed at provision of epidemiological welfare as regards plague]. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2018; (2):45–8. DOI: 10.21055/0370-1069-2018-2-45-48.

6. Eroshenko G.A., Dzhaparova A.K., Oglodin E.G., Al’khova Zh.V., Kukleva L.M., Kuznetsov A.A., Krasnov Ya.M., Abdikarimov S.T., Kutyrev V.V Phylogeny of Yersinia pestis strains belonging to 0.ANT branch, isolated in Tien-Shan and Pamir-Alai in XX–XXI centuries. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2020; (1):76–84. DOI: 10.21055/0370-1069-2020-1-76-84.

7. Kukleva L.M., Dzhaparova A.K., Oglodin E.G., Naryshkina E.A., Krasnov Ya.M., Kuznetsov A.A., Fadeeva A.V., Eroshenko G.A., Berdiev S.K., Kutyrev V.V. [Complex characteristics of Yersinia pestis strains isolated in the Sarydzhaz and Upper-Naryn high-mountain foci in 2019–2020]. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2021; (2):114–22. DOI: 10.21055/0370-1069-2021-2-114-122.

8. Onishchenko G.G., Kutyrev V.V., editors. [Laboratory Diagnostics of Particularly Dangerous Infectious Diseases. Practice Guidelines]. Moscow: JSC “Shiko”; 2013. 560 p.

9. Reed G.H., Kent J.O., Wittwer C.T. High-resolution DNA melting analysis for simple and efficient molecular diag¬ nostics. Pharmacogenomics. 2007; 8(6):597–608. DOI: 10.2217/14622416.8.6.597.

10. Vossen R.H.А.М., Aten E., Roos A., den Dunnen J.T. High-resolution melting analysis (HRMA): more than just sequence variant screening. Hum. Mutat. 2009; 30(6):860–6. DOI: 10.1002/humu.21019.

11. Damgaard P.B., Marchi N., Rasmussen S., Peyrot M., Renaud G., Korneliussen T., Moreno-Mayar J.V., Pedersen M.W., Goldberg A., Usmanova E., Baimukhanov N., Loman V., Hedeager L., Pedersen A.G., Nielsen K., Afanasiev G., Akmatov K., Aldashev A., Alpaslan A., Baimbetov G., Bazaliiskii V.I., Beisenov A., Boldbaatar B., Boldgiv B., Dorzhu C., Ellingvag S., Erdenebaatar D., Dajani R., Dmitriev E., Evdokimov V., Frei K.M., Gromov A., Goryachev A., Hakonarson H., Hegay T., Khacha-tryan Z., Khaskhanov R., Kitov E., Kolbina A., Kubatbek T., Kukushkin A., Kukushkin I., Lau N., Margaryan A., Merkyte I., Mertz I.V., Mertz V.K., Mijiddorj E., Moiyesev V., Mukhtarova G., Nurmukhanbetov B., Orozbekova Z., Panyushkina I., Pieta K., Smrčka V., Shevnina I., Logvin A., Sjögren K.G., Štolcová T., Taravella A.M., Tashbaeva K., Tkachev A., Tulegenov T., Voyakin D., Yepiskoposyan L., Undrakhbold S., Varfolomeev V., Weber A., Wilson Sayres M.A., Kradin N., Allentoft M.E., Orlando L., Nielsen R., Sikora M., Heyer E., Kristiansen K., Willerslev E. 137 ancient human genomes from across the Eurasian steppes. Nature. 2018; 557(7705):369–74. DOI: 10.1038/s41586-018-0094-2.

12. Spyrou M.A., Musralina L., Gnecchi Ruscone G.A., Kocher A., Borbone P.G., Khartanovich V.I., Buzhilova A., Djansugurova L., Bos K.I., Kühnert D., Haak W., Slavin P., Krause J. The source of the Black Death in fourteenth-century central Eurasia. Nature. 2022; 606(7915):718–24. DOI: 10.1038/s41586-022-04800-3.

13. Odinokov G.N., Nikiforov K.A., Kukleva L.M., Krasnov Ya.M., Eroshenko G.A., Kutyrev V.V. [A method for subspecies differentiation of plague agent strains using polymerase chain reaction]. RF Patent No. 2552611, publ. 06/10/2015. Bull. No. 16.

14. Nikiforov K.A., Oglodin E.G., Kukleva L.M., Makashova M.A., Balykova A.N., Eroshenko G.A., Kutyrev V.V. [Construction of an allele-specific real-time PCR system to determine the phylogenetic appurtenance of Yersinia pestis strains]. Biotekhnologiya [Biotechnology]. 2022; 38(3):82–91. DOI: 10.56304/S0234275822030073.

15. Nosov N.Yu., Nikiforov K.A., Eroshenko G.A., Kutyrev V.V. [A method for identifying Yersinia pestis strains of medieval biovar with subsequent differentiation by phylogenetic appurtenance using polymerase chain reaction with hybridization-fluorescent registration of results]. RF Patent No. 2705813, publ. 11/12/2019. Bull. No. 32.

16. Morozov O.A., Nikiforov K.A., Oglodin E.G., Eroshenko G.A., Kutyrev V.V. A method for identifying Yersinia pestis and Yersinia pseudotuberculosis and simultaneous differentiation of Yersinia pestis main and Central Asian subspecies using multiplex PCR. RF Patent No. 2737775, publ. 12/02/2020. Bull. No. 34.

17. Wagner D.M., Klunk J., Harbeck M., Devault A., Waglechner N., Sahl J.W., Enk J., Birdsell D.N., Kuch M., Lumibao C., Poinar D., Pearson T., Fourment M., Golding B., Riehm J.M., Earn D.J., Dewitte S., Rouillard J.M., Grupe G., Wiechmann I., Bliska J.B., Keim P.S., Scholz H.C., Holmes E.C., Poinar H. Yersinia pestis and the plague of Justinian 541–543 AD: a genomic analysis. Lancet. Infect. Dis. 2014; 14(4):319–26. DOI: 10.1016/S1473-3099(13)70323-2.

18. Feldman M., Harbeck M., Keller M., Spyrou M.A., Rott A., Trautmann B., Scholz H.C., Päffgen B., Peters J., McCormick M., Bos K., Herbig A., Krause J. A high-сoverage Yersinia pestis genome from a sixth-century Justinianic plague victim. Mol. Biol. Evol. 2016; 33(11):2911–23. DOI: 10.1093/molbev/msw170.

19. Khvol’son D.A. Nestorian inscriptions from Seven Rivers. In: Rosen V.R., editor. [Notes of the Eastern Branch of the Imperial Russian Archaeological Society. Vol. 1. 1886]. St. Petersburg: Publishing House of the Imperial Academy of Sciences; 1887. P. 217–21.

20. Green M.H. The four Black Deaths. Am. Hist. Rev. 2020; 125(5):1601–31. DOI: 10.1093/ahr/rhaa511.

21. Fancy N., Green M.H. Plague and the fall of Baghdad (1258). Med. Hist. 2021; 65(2):157–77. DOI: https://doi.org/10.1017/mdh.2021.3.

22. Kol’chenko V.A. [Pripishpek (Kara-Dzhigach) medieval Christian cemetery according to archival documents about excavations in 1886]. In: [Religions of Kazakhstan and Central Asia across the Great Silk Road: Materials of the International Scientific and Practical Conference (Almaty, June 12–13, 2017)]. Almaty; 2017. P. 167–79.

23. Kol’chenko V.A. [Christian cemetery of the Mongol era at the site of Burana (according to archival documents about excavations in 1886)]. In: Bocharov S.G., Sitdikov A.G., editors. [Genoese Khazaria and the Golden Horde]. Kazan; Kishinev; 2019. Vol. 2. P. 209–21.