Tularemia is a highly dangerous zoonotic infection due to the bacteria Francisella tularensis. Low genetic diversity promoted the use of polymorphic tandem repeats (MLVA) as first-line assay for genetic description. Whole genome sequencing (WGS) is becoming increasingly accessible, opening the perspective of a time when WGS might become the universal genotyping assay. The main goal of this study was to describe F. tularensis strains circulating in Kazakhstan based on WGS data and develop a MLVA assay compatible with in vitro and in silico analysis. In vitro MLVA genotyping and WGS were performed for the vaccine strain and for 38 strains isolated in Kazakhstan from natural water bodies, ticks, rodents, carnivores, and from one migratory bird, an Isabellina wheatear captured in a rodent burrow. The two genotyping approaches were congruent and allowed to attribute all strains to two F. tularensis holarctica lineages, B.4 and B.12. The seven tandem repeats polymorphic in the investigated strain collection could be typed in a single multiplex PCR assay. Identical MLVA genotypes were produced by in vitro and in silico analysis, demonstrating full compatibility between the two approaches. The strains from Kazakhstan were compared to all publicly available WGS data of worldwide origin by whole genome SNP (wgSNP) analysis. Genotypes differing at a single SNP position were collected within a time interval of more than fifty years, from locations separated from each other by more than one thousand kilometers, supporting a role for migratory birds in the worldwide spread of the bacteria.

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.

Background and Aim: Salmonella Abortusequi is a significant etiological agent of equine abortions, yet limited genomic data exist, particularly in Central Asia. This study aimed to perform the first genome-wide characterization and phylogenetic analysis of three S. Abortusequi strains isolated from equine abortions in different regions of Kazakhstan. Materials and Methods: Whole-genome sequencing was conducted on three isolates using the Illumina MiSeq platform. Genomic assemblies were annotated using SPAdes and Prokka, while phenotypic traits were predicted through BioNumerics. Antimicrobial resistance genes, virulence factors, and prophage elements were identified using established databases. Phylogenetic relationships were examined through whole-genome single-nucleotide polymorphism (wgSNP) analysis against a global panel of S. Abortusequi and related serovars. Results: All isolates displayed high genomic similarity and were classified as Salmonella enterica subsp. enterica serovar Abortusequi with an antigenic profile of 4:a:e,n,x. Twelve Salmonella pathogenicity islands and three prophages were identified, with ST64B present in all isolates. The ac(6’)-Iaa gene, which confers resistance to aminoglycosides, was detected in all strains. Each genome encoded 101–109 virulence factors, with 94 conserved across isolates. wgSNP analysis confirmed close phylogenetic clustering of the Kazakh strains, with regional variation between northern and southern isolates. Prophage-associated virulence elements, particularly virulence factor protein (SseK), were also documented. Conclusion: This study reveals the genetic uniformity and virulence potential of S. Abortusequi strains circulating in Kazakhstan. The presence of conserved resistance and virulence genes, including prophage-encoded elements, underscores the pathogenic risk posed by these isolates. These findings contribute valuable genomic data for surveillance, diagnosis, and control of salmonellosis in equine populations. Despite the limited sample size, the study establishes a foundation for future genomic epidemiological studies and targeted disease mitigation strategies. Keywords: Antimicrobial resistance, equine abortion, Kazakhstan, pathogenicity islands, phylogenomics, Salmonella Abortusequi, whole-genome sequencing.

Background and Aim. Abortion associated with equine salmonellosis is an infectious disease that significantly affects the productivity of herd-based horse breeding in the Republic of Kazakhstan. The disease is characterized by late term abortions and is associated with considerable economic losses in the agricultural sector. The aim of this study was to detect Salmonella DNA using molecular genetic methods. Materials and Methods. The study was based on pathological material collected from horses in nine regions of Kazakhstan during the period from 2023 to 2025. Results. PCR analysis revealed that the proportion of positive samples ranged from 25% to 33.3%, depending on the year. Enzyme-linked immunosorbent assay (ELISA) of 309 equine serum samples detected antibodies to Salmonella abortus equi in 29 cases (10.6%). Microbiological investigations using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) confirmed the presence of Salmonella spp., as well as opportunistic microorganisms such as Enterobacter hormaechei, Pantoea agglomerans, and Escherichia hermannii. Conclusion. The obtained results confirm the circulation of the pathogen among horses and emphasize the need for ongoing epizootiological surveillance. Furthermore, they highlight the importance of continued molecular and genetic studies, including whole genome sequencing, to enhance understanding of the disease’s epidemiology and to optimize preventive measures.