The thesis is devoted to the phylogenetic analysis of Ukrainian Bacillus anthracis isolates, as well as to the improvement of methods for the detection of anthrax genome and plasmids via polymerase chain reaction.
In order to search for isolates for studies, 11 field expeditions have been conducted to anthrax animal burial sites in Kharkiv, Sumy and Mykolaiv oblasts (regions), where soil samples were taken for further isolation of B. anthracis spores. Besides, DNA samples of Ukrainian anthrax isolates obtained in previous years by regional laboratory centers from various sources (from infected people and from environmental samples) were used for phylogenetic analysis.
B. anthracis isolate was obtained from 50 cm depth at anthrax grave site nearby Koviagy village, Valky rayon (district), Kharkiv oblast (region).
For the first time, we carried out genotyping and full-genome sequencing of DNA of some other Ukrainian isolates and vaccine strains, including above-mentioned isolate. Obtained results had shown the presence of heterogenic population of B. anthracis in Ukraine, consisting of at least three different canSNP groups of worldwide-dominating A-branch of this pathogen. The above-mentioned soil isolate represents one of these three groups. It is closely related with STI vaccine strain. The other phylogenetic group, namely A.Br.008/009, might be autochthonic. This group is characterized by the occurrence of closely related strains in South-Eastern European countries, which are geographically close to Ukraine. The results of full-genome sequencing of soil isolate from Mykolaiv oblast show the appearance of another, third canSNP group, namely Vollum, which is untypical for Ukraine. This group is widely spread in Asian regions (namely in Pakistan). Sequencing of other isolates from Sumy and Kherson oblasts has shown that they are closely-related to Japanese isolates.
Also, we prepared recombinant positive controls for the detection of pagA, pXO1 plasmid marker, capC, pXO2 plasmid marker and gyrA, Bacillus multispecies gene. Their workability for using at diagnostic veterinary laboratories has been proved via qPCR and sequencing.
As a result of qPCR method’s optimization, we proved that it is specific and reliable for the detection of pagA and capC genetic markers. Besides, this validation procedure allowed to improve robustness, repeatability and sensitivity of this method.
‘Anthrax-DNA-test’, test-kit for the detection of anthrax DNA via polymerase chain reaction was created. It includes above-mentioned recombinant positive controls for the detection of anthrax plasmid markers, as well as positive control for the detection of B. anthracis dhp61 chromosomal marker. It is not inferior in specificity, sensitivity and repeatability from the method of anthrax detection recommended by OIE, as well as foreign analogues.
