The aim of the study was to investigate the prevalence of minor cattle infections (leukemia, bovine immunodeficiency and spumavirus infection) using the polymerase chain reaction (PCR). Blood samples were collected from cows in conditionally leukemia-free farms in ten regions of Ukraine to determine the presence of these infections. The samples were examined via classical PCR to detect the genetic material of the specific fragment of the ENV gene of the leukemia virus using BLV-env-3/BLV-env-4 primers recommended by the OIE. To identify the proviral DNA of bovine foamy virus (BFV), primers Int 1-Int 2 and Int 3-Int 4 were used, and for the detection of bovine immunodeficiency virus (BIV) proviral DNA, a pair of primers RT_+(–) flanking the conservative domain of reverse transcriptase and a pair of primers flanking the pol gene of the BIV were selected. The situation concerning leukemia is most severe in Sumy and Kharkiv regions. A significant percentage of animals carrying the foamy virus was observed in farms in Kirovohrad, Kherson, Donetsk, and Kharkiv regions. Moreover, genetic material of the immunodeficiency virus was found in samples from Kirovohrad, Donetsk, and Kherson regions. These results indicate a significant prevalence of minor infections among cattle in Ukraine due to a lack of awareness among livestock workers, highlighting the necessity for comprehensive sanitary and preventive measures

This paper represents preliminary trials of the ‘Anthrax-DNA-test’, diagnostical tool for the detection of anthrax DNA. It includes recombinant positive controls p-pagA-TZ57R/T and p-capC-TZ57R/T for the detection of anthrax plasmid markers, as well as p-dhp61-CR2.1-TOPO, positive control for the detection of Bacillus anthracis chromosomal marker. Besides, three mixtures of primers and probes for the detection of each genetic marker (dhp61, pagA, and capC) and ready-to-use ‘RT-PCR МаsterМіx’ PCR diluent were also included. Concentrations of MgCl2 and Taq-polymerase obtained during qPCR validation procedure were considered when preparing the diluent. To determine specificity, qPCR was conducted with heterological panel of DNA of pathogenic bacteria and viruses causing diseases with similar to anthrax clinical signs. To determine repeatability of the results when using ‘Anthrax-DNA-test’ PCR test kit, samples were studied twice. The sensibility of the kit was analyzed by serial dilutions of p-dhp61-CR2.1-TOPO, p-pagA-TZ57R/T and p-capC-TZ57R/T plasmid DNAs containing fragments of anthrax chromosome and plasmids. To compare the tool’s ability to identify anthrax DNA, classical PCR was carried out using ANT-PA_F/R and ANT-CAP_F/R primers recommended by OIE for the detection of pXO1 and pXO2 plasmid DNA. Sensitivity testing has shown that the test kit is able to identify all positive samples. It has been found that the diagnostics tool detects anthrax DNA in recombinant positive control samples containing B. anthracis chromosomal and plasmid DNA fragments in serial dilutions from 1:100 to 1:1,000 with Ct values of 25.29–34.70. The specificity of this diagnostic tool is proved by the absence of Ct in heterological samples. Besides, repeatability of trial results has been found, which is proved by complete congruence in duplicates with each of the tested sample

Aim. The aim of this study was to screen soil samples of 17 anthrax burial sites in Eastern and Southern Ukraine for the presence of B. anthracis. Methods. Soil samples were collected from anthrax grave sites located in Kharkiv, Sumy and Mykolaiv regions (diseased animals dated from 1946 to 2003). Isolation of B. anthracis from collected soil samples was performed with the GABRI method. From single colonies without hemolysis, that were inactivated with peracetic acid- containing 2 % Terralin PAA solution, DNA was extracted and analyzed by qPCR for the presence of chromosomal marker dhp61, as well as the markers pagA and capC located on virulence plasmids pXO1 and pXO2, respectively. Results. Eleven fi eld trips were conducted from July, 2016 to October, 2018 in which 369 soil samples from 17 burial sites in Kharkiv, Sumy and Mykolaiv oblasts were collected from different depths of presumed anthrax carcass sites. In most cases (12 out of 17 cases), the current status of these burial sites was deteriorated and not prop- erly accounted for. It was possible to obrain viable B. anthracis isolate was obtained from 50 cm depth at the grave site near Koviagy village, Valky district, Kharkiv region (49.92373°N, 35.48951°E). This isolate was named KhR/ VD/Kov2-2-05-3 and deposited in the Collection of Animal Infectious Pathogens of the National Scientifi c Center “Institute of Experimental and Clinical Veterinary Medicine”, Kharkiv, Ukraine. The contamination level of soil at the isolation site reached about 10 4 CFU per g as determined by plate counting. qPCR analysis of this isolate identi- fi ed both the dhp61 B. anthracis chromosomal and the pagA virulence plasmid marker. However, the plasmid pXO2 marker, required for capsule-formation could not be detected. Conclusions. The anthrax burial sites were created between the 1920s and 1960s, however, only approximate locations could be found and demarcated. In most cases the status of the sites was unsuitable for sampling. Nevertheless, isolation of B. anthracis in one case in the Valky district shows that old anthrax burial sites (13.500 exist in Ukraine) still pose a risk as potential source of the infection and therefore require more attention and surveillance, for which a surveillance plan will be developed.

This paper represents qPCR validation results for the detection of Bacillus anthracis pagA pXO1 plasmid marker. The aim of the work was to transfer, implement and validate anthrax specific pagA qPCR assay for the detection of pagA, the genetic marker of the pXO1 plasmid of Bacillus anthracis. qPCR was conducted using the Applied Biosystems Fast 7500 Real-time PCR system including Applied Biosystem specific reagents (AmpliTaq Gold). Anthrax pXO1 pagA primers (pagA_forward, pagA_reverse) and TaqMan pagA probe. Data analysis and statistical calculations were performed using Microsoft Excel. The limit of detection (probit analysis) was calculated using the Statgraphics software. Robustness of qPCR was adjusted by optimization of amplification parameters (annealing temperature) and concentration of reaction components (MgCl2, primers, probe and Taq polymerase). In order to test the repeatability and precision of the qPCR assay after optimization, the variation within the experiment (Intra-assay variability) and between several independent experiments (Inter-assay variability) was evaluated. Probit analysis with serial dilutions of positive control with five replicates per dilution was carried out to define the 95% limit of detection (LOD). To determine if the CT value correlates with the amount of template DNA, the linearity of qPCR was analyzed. The standard curve was generated and the linear regression line and the coefficient of correlation (R2) were calculated. To define the ability to detect sequence of interest (sensitivity), we tested mixed panel of Bacillus anthracis DNAs. As the result, pagA marker could be detected in all tested strains . To find out the specificity of our assay, we also tested DNA of various strains of B. cereus, B. thuringiensis, B. mycoides, and B. globigii (potential cross-reacting organisms) as well as DNA samples of various pathogenic bacteria and viruses which cause similar clinical symptoms as anthrax (differential diagnosis relevant organisms).

ObjectiveDue to the lack of information about the phylogenetic origins of Ukrainian Bacillus anthracis strains,the goal of this work was to make phylogenetic analysis of Ukrainian isolates obtained from various sources (soil, clinical material from infected humans and animal products) for better understanding of phylogenetic origins of this pathogen in Ukraine and Eastern Europe.IntroductionAnthrax is a widely spread zoonotic disease with natural transmissive cycle involving wildlife, livestock and humans [1]. It is caused by Bacillus anthracis, a highly pathogenic gram-positive, spore-producing bacterium, which poses a serious threat to public and animal health due to its mortality both for animals and for humans [2, 3, 4]. The ability of B. anthracis spores to remain viable in soils for decades enables their isolation from freely accessible environment [5]. This unique feature to form highly resistant spores in the environment plays a major role in the ecology and evolution of this pathogen [6]. During the spore phase, evolution is greatly reduced in rate, which limits the amount of genetic diversity found among isolates of this species [1]. All these factors demonstrate the need for reliable anthrax diagnosis and trace-back methods. This comprises bio forensic capabilities including state-of-the-art methods for accurate genotyping of B. anthracis strains.Methods23 thermolysates of B. anthracis broth cultures isolated from various sources (vesicles from eleven different people infected with cutaneous anthrax when disease’s sporadic outbreaks were detected in Ukraine in 1963-2002, as well as two samples from sheep wool, and eight soil samples) were obtained from the Central Epidemiological Station (Kyiv, Ukraine), as well as from I.I. Mechnikov Ukrainian Scientific and Research Anti-plaque Institute (Odessa, Ukraine). These anthrax cultures were confirmed with classical microbiological methods (microscopy, cultivation on solid and liquid media), “string of pearls” reaction, and using bioassay on living white mice (the mortality was observed two days after subcutaneous injection of 0,2-0,5 ml of cells’ suspension). All these tests were carried out at the institutions where samples were obtained. Besides, one B. anthracis isolate was cultivated from soil sample of an animal grave site nearby Koviagy village, Valky district, Kharkiv region. All samples were analyzed at the Bundeswehr Institute of Microbiology (Munich, Germany). To confirm the presence of the anthrax genome and plasmids, we isolated genomic DNA (gDNA) from thermolysates and studied the presence of the genomic marker dhp61 as well as the plasmid specific marker pagA (pXO1) and capC (pXO2) using qPCR. Quality of the isolated gDNA was tested using the Agilent bioanalyzer. To characterize regional and global phylogeographic patterns of these strains, canonical Single Nucleotide Polymorphisms analysis (canSNP) was conducted using high resolution melt (HRM). Three thermolysates of broth cultures isolated and soil sample isolated from animal grave site in Kharkiv region were analyzed using NewSeq Full genome sequencing.ResultsB. anthracis chromosomal DNA-marker dhp61 as well as pXO1 marker pagA and pXO2 plasmid marker capC could be detected in all thermolysates. However, the soil isolate from the Koviagy grave site was positive for dhp61 but contained only the pXO1 plasmid. The Bioanalyzer assay revealed that only 6 out of the 23 thermolysates had good enough DNA quality to be sequenced. So far only genomes of thermolysates of soil samples from Mykolaiv and Sumy regions, the thermolysate of sick patient's vesicle from Kherson region as well as the soil sample from the animal grave site in Kharkiv region have been sequenced. For the residual 3 thermolysates the full genome analysis is still in progress. The sequencing results showed that the B. anthracis strain isolated from Mykolaiv soil sample belongs to the Vollum linage group and other thermolysates from Sumy and Kherson regions are closely clustering with isolates from Japan. Thus, human isolate from Kherson region is clustering with the Japanese isolate BA104 which was obtained from pig during sporadic anthrax incident in 1982 and soil isolate from Sumy region is clustering with the BA 103 isolate which was obtained from beef cattle in Japan in 1991. In contrast, we analyzed the genomic sequence of the pXO2-negative isolate from grave site in Kharkiv region using BioNumerics software and found that it has high similarity to STI strain.ConclusionsThe infrequent sporadic occurrence of anthrax in the country of Ukraine is likely caused by a heterogeneous population of B. anthracis. The found STI strain in the grave site of Kharkiv region is probably an environmental recovery of the Russian anthrax live vaccine which was commonly used for vaccination of animals in the former Soviet Union The sequencing result of the soil isolate from Mykolaiv region indicates the occurrence of another canSNP group, the Vollum group, which is quite untypical for Ukraine. The latter is mainly prevalent in the Asian regions (namely Pakistan) and therefore might have been introduced to Ukraine over the silk road. Other two thermolysates from Sumy and Kherson regions also showed unexpected results clustering with Japanese isolates. The further research of Ukrainian B. anthracis isolates will allow us to expand our knowledge about the population structure and evolution of anthrax in Ukraine.References1. Van Ert MN, Easterday WR, Huynh LY, Okinaka RT, Hugh-Jones ME, Ravel J, et al. (2007) Global Genetic Population Structure of Bacillus anthracis. PLoS ONE 2(5);2. Freidlander, A. M. 1997. Anthrax, p. 467–478. In F. R. Sidell, E. T. Takafuji, and D. R. Franz (ed.), Medical aspects of chemical and biological warfare. Office of the Surgeon General, Washington, D.C.3. Hoffmaster AR, Fitzgerald CC, Ribot E, Mayer LW, Popovic T (2002) Molecular subtyping of Bacillus anthracis and the 2001 bioterrorism-associated anthrax outbreak, United States. Emerg Infect Dis 8: 1111–1116.4. Keim P, Van Ert MN, Pearson T, Vogler AJ, Huynh LY, et al. (2004) Anthrax molecular epidemiology and forensics: using the appropriate marker for different evolutionary scales. Infect Genet Evol 4: 205–213.5. Eitzen, E. M. 1997. Use of biological weapons, p. 437–450. In F. R. Sidell, E. T. Takafuji, and D. R. Franz (ed.), Medical aspects of chemical and biological warfare. Office of the Surgeon General, Washington, D.C.6. Biloivan O, Duerr A, Schwarz J, Grass G, Arefiev V, Solodiankin O, Stegniy B, Gerilovych A (2018) Phylogenetic analysis of Ukrainian Bacillus anthracis strains. Third Annual BTRP Ukraine Regional One Health Research Symposium, abstract directory: 122.

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