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

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

doi:10.15407/agrisp7.03.003

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.

Highlights

Anthrax is a zoonotic disease to which mainly grazing herbivores, and omnivores, carnivores and human are susceptible
369 soil samples were collected of 17 locations only (1 in Sumy, 4 in Mykolaiv and 12 in Kharkiv regions)
According to Hugh Jones & Blackburn (2009), B. anthracis spores longer stay viable in humus soils rich with calcium, phosphorus and magnesium, than in sands or loams

Summary

Introduction

Anthrax is a zoonotic disease to which mainly grazing herbivores, and omnivores, carnivores and human are susceptible. Under adverse conditions the causative pathogen Bacillus anthracis is able to form spores that may remain viable in the environment, especially in soil, for many decades Due to this biological feature and its high virulence, this pathogen is still of great scientific and epidemiological importance in many areas of the world. More than a million (mainly wild) animals and about 20,000 people in 82 countries around the world get infected with anthrax (Logvin et al, 2017). This pathogen is easy to multiply and has a high recalcitrance in the environment. Laboratory produced anthrax spores of pathogenic strains represent a potential threat related to bioterrorism, as they are easy to produce and store (Hoffmaster et al, 2001; Keim et al, 2004; Eitzen et al, 1997)

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