First record of the human infection of Brucella melitensis in Kyrgyzstan: evidence from whole-genome sequencing-based analysis.

BACKGROUND: Brucellosis, a zoonosis mainly transmitted by consumption of unpasteurized dairy products as well as direct contact with infected animals, is endemic in Kyrgyzstan. However, Brucella species in humans have not been investigated and the origin of the disease remains poorly known in wide parts of Сentral Asia. Thus, molecular characterization of the circulating strains is a critical first step in understanding Brucella diversity in the country. METHODS: In this study, isolates were collected from patients with suspected brucellosis from different regions in Kyrgyzstan between 2019 and 2020. The detection and identification of Brucella was carried out by Bruce-ladder PCR. Next generation sequencing was used to sequence the 89 Brucella isolates, which were genotyped by cgSNP and cgMLST to identify epidemiological connection between Brucella isolates as well as placing them in the context of the global Brucella phylogeny. RESULTS: The Brucella strains isolated from all regions of Kyrgyzstan were identified as B. melitensis. Based on cgSNP analysis, 18 sequence types were differentiated. The highest numbers of different sequence types were found in Batken (n = 8), Osh (n = 8) and Jalal-Abad (n = 6) oblasts. According to cgSNP and cgMLST analyses, different B. melitensis lineages circulate in Kyrgyzstan, all of them belonging to the Eastern Mediterranean group of the global Brucella phylogeny with the highest similarity to strains from Turkmenistan, Iran and Turkey. CONCLUSION: In the present study, B. melitensis was identified as a causative agent of human brucellosis in Kyrgyzstan and different lineages could be identified. Since this study focused on isolates of human origin, the identity of Brucella species and lineages circulating among animal populations remains elusive. Implementing culture techniques and use of most recent molecular, bioinformatic and epidemiological tools are needed to set up a One Health approach to combat brucellosis in Kyrgyzstan. Further, other Сentral Asian countries need to take part in this effort as brucellosis is a transboundary disease in these regions.

Marmots and Yersinia pestis Strains in Two Plague Endemic Areas of Tien Shan Mountains.

The main purpose of this study was to clarify the role of gray marmots (Marmota baibacina) in the long-term maintenance of highly virulent strains of Yersinia pestis in two plague endemic foci of the Tien Shan Mountains in Kyrgyzstan. We present data from regular observations of populations of M. baibacina and small rodents cohabiting with marmots in the mountainous grasslands of the Sari-Dzhas (east of Issyk-Kul Lake) and the Upper-Naryn (south of Issyk-Kul Lake) natural foci. During 2012-2017, an abundance of marmots and their ectoparasites (fleas and ticks) was significantly higher in Upper-Naryn comparing to Sari-Dzhas, although there were no differences in a number and diversity of small rodents cohabiting with marmots. The plague bacterium was detected either in marmots or in their ectoparasites collected during 4 of 6 years of observation in Sari-Dzhas and during 2 of 4 years of observation in Upper-Naryn. Plague was found in three sectors situated closely to each other in Sari-Dzhas and in 1 of 8 repeatedly surveyed sectors in Upper-Naryn. During 6 years, we isolated 9 strains of Y. pestis from marmots, two from their fleas Oropsylla silantiewi, one from an unidentified tick, and one from the gray hamster (Cricetulus migratorius). All plague strains isolated from the rodents and their ectoparasites in this study were similar to Antiqua biovar specific for marmots. The results indicate that plague can circulate continuously in the Tien Shan Mountains in populations of gray marmots and their ectoparasites with a facultative involvement of other rodent species after significant changes in rodent communities that happened in Kyrgyzstan during the previous two decades. The simultaneous field survey of two natural foci of plague, Sari-Dzhas, and Upper-Naryn, would be important for further analysis of circulation of Y. pestis strains belonging to Antiqua biovar in the Tien Shan Mountains.

Current Status of the Sari-Dzhas Natural Focus of Plague, Kyrgyzstan: Epizootic Activity and Marmot Population.

The Sari-Dzhas natural mountain focus of plague with an area of 5000 sq. km is located mainly in Kyrgyzstan. This enzootic area belongs to a group of Tien-Shan mountain plague foci and crosses the boundaries of Kazakhstan, Kyrgyzstan, and China. After collapse of Soviet Union, this area was not investigated fully, thence we have estimated current status of hosts and vectors comparing to 1977 in the Sari-Dzhas focus. For period 1977-2014, the number of the gray marmot (Marmota baibacina) as main host of plague has decreased twice due to regular chemical suppression of focal area, but now there is a tendency for population recovery. If negative impact of human activity (hunting, livestock, and tourism) will be minimized, the population density should rise in the near future, related to high reproduction score of animals. Significant increase of number of narrow-skulled vole (Microtus gregalis) observed on focus area in 1977-2017 can effort a risk of pathogen transmission in cross-populations of marmots provided by recovery of vector number between different hosts. For whole observation period in the Sari-Dzhas focus were described 33 species of marmot fleas with dominated Oropsylla silantiewi (67% from whole amount of fleas) and Radinopsilla li ventricosa (6.5%). Deep dustation of marmot burrows by 10% dichlorodiphenyltrichloroethane (DDT) conducted in 1980s caused significant decrease of ectoparasite abundance: the number of specific fleas is strongly diminished, while recovery of ticks and lice number is faster. At the same time, Yersinia pestis constantly circulates in the focal area, which is comfortable for interacting of human populations and pathogen hosts. Last plague epizootic was recorded in 1983 and recent outbreaks among gray marmot and its vectors with varying intensity are registered again starting from 2012. Thus, the Sari-Dzhas natural plague focus remains active with all links of plague epizootic cycle (marmot-vector-rodent) and needs strong monitoring every year.

Yersinia pestis strains of ancient phylogenetic branch 0.ANT are widely spread in the high-mountain plague foci of Kyrgyzstan.

Fifty six Yersinia pestis strains, isolated over the period of more than 50 years in three high-mountain foci of Kyrgyzstan (Tien Shan, Alai, and Talas), have been characterized by means of PCR and single nucleotide polymorphism (SNP) typing methods. Seven of these strains were also characterized by means of whole genome sequencing and genome-wide SNP phylogenetic analysis. It was found that forty two strains belong to 0.ANT2, 0.ANT3 and 0.ANT5 phylogenetic branches. From these, strains of 0.ANT2 and 0.ANT3 branches were earlier detected in China only, whereas 0.ANT5 phylogenetic branch was identified for Y. pestis phylogeny for the first time. According to the results of genome-wide SNP analysis, 0.ANT5 strains are ones of the most closely related to Y. pestis strain responsible for the Justinianic Plague. We have also found out that four of the studied strains belong to the phylogenetic branch 2.MED1, and ten strains from Talas high-mountain focus belong to the phylogenetic branch 0.PE4 (sub-branch 0.PE4t). Established diversity of Y. pestis strains and extensive dissemination of the strains pertaining to the 0.ANT branch confirm the antiquity of the mentioned above plague foci and suggest that strains of the 0.ANT branch, which serve as precursors for all highly virulent Y. pestis strains, had their origin in the Tien Shan mountains.