Screening of possibly anthrax-contaminated burial sites in eastern and southern Ukraine

O. V. Biloivan; B. T. Stegniy; A. P. Gerilovych; V. L. Arefiev; R. Wölfel; J. Schwarz; C. Popp; G. Grass

doi:10.15407/agrisp7.03.003

O. V. Biloivan NSC «Institute of Experimental and Clinical Veterinary Medicine of NAAS», 83 Pushkinska str., Kharkiv, Ukraine, 61023
B. T. Stegniy «Institute of Experimental and Clinical Veterinary Medicine of NAAS», 83 Pushkinska str., Kharkiv, Ukraine, 61023
A. P. Gerilovych «Institute of Experimental and Clinical Veterinary Medicine of NAAS», 83 Pushkinska str., Kharkiv, Ukraine, 61023
V. L. Arefiev «Institute of Experimental and Clinical Veterinary Medicine of NAAS», 83 Pushkinska str., Kharkiv, Ukraine, 61023
R. Wölfel Bundeswehr Institute of Microbiology, Neuherbergstr. 11, Munich, Germany, 80937
J. Schwarz Bundeswehr Institute of Microbiology, Neuherbergstr. 11, Munich, Germany, 80937
C. Popp Bundeswehr Institute of Microbiology, Neuherbergstr. 11, Munich, Germany, 80937
G. Grass Bundeswehr Institute of Microbiology, Neuherbergstr. 11, Munich, Germany, 80937

DOI: https://doi.org/10.15407/agrisp7.03.003

Keywords: anthrax, B. anthracis, animal burial sites, soil, spores, polymerase chain reaction, DNA

Abstract

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.

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