Tularemia is an acute febrile disease caused by the Gram-negative bacillus Francisella tularensis. Based on genetic and phenotypic characteristics, three subspecies are distinguished: tularensis, holarctica, and mediasiatica. F. tularensis subsp. mediasiatica remains the least studied subspecies. Over the past decade, new foci of distribution of F. tularensis subsp. mediasiatica have been discovered in Russia (Siberia), expanding the possible distribution area by thousands of kilometers. This article provides whole genome single nucleotide polymorphism (wgSNP) and polymorphic tandem repeats (MLVA) analyses of 28 mediasiatica strains isolated between 1965 and 2004 in Kazakhstan. Despite high genetic homogeneity, MLVA with eleven loci (MLVA11) demonstrates a high discriminatory ability (diversity index, 0.9497). The topological structure of the trees based on wgSNP and MLVA is not comparable; however, clustering remains congruent for most outbreaks, with the exception of two strains from one outbreak that are identical in terms of wgSNP but differ at three tandem repeat loci. Based on wgSNP, the strains are assigned to one of the three currently known mediasiatica sublineages, lineage M.I, together with other historical strains maintained in collections in Russia and Sweden. wgSNP shows limited previously unknown genetic diversity, with the M.I lineage size being only 118 SNPs. The wgSNP genotype is not strongly correlated with year and place of isolation.

Tularemia is an acute infectious disease classified as a natural focal infection, requiring continuous monitoring of both human and animal morbidity, as well as tracking of pathogen circulation in natural reservoirs and vectors. These efforts are essential for a comprehensive prevention and containment strategy. The causative agent, Francisella tularensis, comprises three subspecies—tularensis, holarctica, and mediasiatica—which differ in their geographic distribution and virulence. The ability to directly detect the pathogen and differentiate between subspecies has enhanced diagnostics and allowed a more accurate identification of circulation areas. Real-time PCR protocols for identification of F. tularensis subspecies tularensis and holarctica have been developed, utilizing specific primers and probes that target unique genomic regions. In this study, we present the development of a new real-time PCR assay for the detection of Francisella spp. and differentiation of F. tularensis subsp. mediasiatica. The specificity of the assay was tested on DNA from 86 bacterial species across 31 families unrelated to Francisella spp., as well as on DNA collections of F. tularensis subsp. mediasiatica and F. tularensis subsp. holarctica. The limit of detection (LOD95%) for real-time PCR in detecting Francisella spp. was 0.297 fg (0.145 genomic equivalents, GE) for holarctica DNA and 0.733 fg (0.358 GE) for mediasiatica DNA. The LOD95% for subspecies differential identification of mediasiatica was 8.156 fg (3.979, GE). The high sensitivity and specificity of these developed protocols enable direct detection of pathogens in biological and environmental samples, thereby improving the efficiency of tularemia surveillance in Kazakhstan.

Hemorrhagic fever with renal syndrome (HFRS) is a zoonotic disease associated with orthohantaviruses maintained in natural rodent hosts. This study assessed the detection and spatial distribution of hantavirus RNA among small mammals in the West Kazakhstan Region in 2025 and compared rodent surveillance findings with human HFRS occurrence. An observational cross-sectional analytical study was conducted in July and September 2025 in four districts. A total of 343 small mammals were captured and morphologically identified. Lung tissue samples from 329 animals collected in September were examined in 123 pooled testing units, whereas 14 animals collected in July were tested individually. Thus, 137 testing units representing 343 animals were evaluated by real-time and conventional RT-PCR. The minimum infection rate (MIR) was calculated as a descriptive minimum estimate, and a pooled-testing likelihood model was used to account for variation in testing-unit size. Hantavirus RNA was detected in 47/137 testing units, including 45 positive pooled units and two individually positive specimens. Based on 343 individual animals, the descriptive MIR was 13.7%. The model-based individual-level infection probability was estimated at 18.0% (95% profile-likelihood CI: 13.5–23.1%). Positive testing units were observed among the Ural field mouse (Apodemus uralensis), bank vole (Myodes glareolus), and common vole (Microtus arvalis). At the district level, the descriptive MIR ranged from 11.6% in Burlinsky to 19.0% in Chingirlauisky. Sampling sites overlapped spatially with settlements reporting human HFRS cases, including Utvinka, Daryinsk, and Rubezhinskoye; however, spatial coincidence does not establish direct transmission.