You are here

Determination of effi ciency of the methods for isolating Bacillus anthracis pathogen spores from soil

Diseases of animals and people with anthrax continues to appear in traditionally endemic regions, indicating a real threat of disease in any territory: in Europe, Asia and other regions of the globe. Spores of the causative agent of anthrax are very stable in the environment. It is known that they remain viable for up to 300 years. Under favorable conditions, activation (mobilization of metabolic processes) of spores occurs within 1–2 minutes, followed by initiation and germination (20–30 minutes) and growth into a vegetative cell (60–90 minutes). Zoonotic anthrax disease is relevant not only for Ukraine, but also for most countries in Europe and around the world, as the incidence remains high, thousands of deaths are registered annually, large sums of citizens and states are spent on the treatment of patients, and producers of livestock products bear considerable economic losses due to the death of animals and carrying out anti-epizootic, anti-epidemiological and preventive measures. The article presents the results of determining the eff ectiveness of methods for isolating the spores of the causative agent of the strain Bacillus anthracis UA–07 from the soil. It was found that using the method using Tween-80, 1 % serum albumin in phosphate-buff ered saline and PLET agar, it was possible to increase the excretion of pathogen spores from the soil by 16.8 % (p <0.001). At the same time, it should be noted that with a slight contamination of the soil or material under study, there is probably a problem of obtaining an unreliable study result. According to our advanced methodology, only 2.5 g of soil is needed for the study, whereas according to the method № 1 – 60 g, according to the method № 2 and № 3 – 95, № 4 – 10 g. higher spin speed, uses 100% ethyl alcohol, which destroys the soil's vegetative and some spore like microfl ora (except for spores of Bas. anthracis). 

Key words: anthrax, spores, Bacillus anthracis, animals, soil, distribution, methodology.


1. Gómez, J.P.,  Nekorchuk, D.M., Liang Mao., Blackburn J.K. (2018). Decoupling environmental eff ects and host population dynamics for anthrax, a classic reservoirdriven disease. Journal of PLOS one. pp. 1–20. Available at:

2. Morris, L., Proffi  tt, K., Asher, V., Blackburn, J. (2015). Elk resource selection and implication s for anthrax management in Montana. Journal of wildlife management. no. 80, pp. 235–244.

3. Blackburn, J., Asher, V., Stokke, S., Hunter, D. (2014). Dances with Anthrax: Wolves (Canis lupus) Kill Anthrax Bacteremic plains bison in Southwestern Montana. Journal of Wildlife disease. no. 50, pp. 393–396.

4. Salb, A., Stephen, C., Ribble, C., Elkin, B. (2014). Descri ptive epidemiology of detected anthrax outbreaks in wild wood bison (Bison bison at habascae) in northern Canada, 1962–2008. Journal of Wildlife Disease. pp. 459–468.

5. Blackbun, J.K., Van, Ert M., Mullins, J.C., Hadfi eld, T.L., Hugh-Jones, M.E. (2018). The necrophagous fl y anthrax transmits ion pathway: empirical and genetic evidence from wildlife epizootics. Vector-Borne and Zoonotic Disease 2014. Journal of PLOS one. 20, 14 (8), pp. 576– 583.

6. Driciru, M., Rwego, I.B., Asiimwe, B., Pelican, K. (2018). Spatio-temporal epidemiology of anthrax in Hippopotamus amphibious in Queen Elizabeth Protected Area, Uganda. Journal of PLOS one. 13 (11). 21 р. Available at: Spatio-temporal_epidemiology_of_anthrax_in_ Hippopotamus_amphibious_in_Queen.

7. Lepheana, R.J., Oguttu, J.W., Nenene, Q. (2018). Temporal patterns of anthrax outbreaks among livestock in Lesotho, 2005–2016. Journal of public health. pp. 1–12.

8. Kracalik, I., Malania, L., Broladze, M., Navdarashvili, A., Imnadze, P., Ryan, S.J., Blackburn, J.K. (2017). Changing livestock vaccination policy alters the epidemiology of human anthrax, Georgia, 2000–2013. Journal vaccine. Vol. 35, Issue 46, pp. 6283–6289.

9. Rublenko, І.O., Skripnik, V.G. (2016). Analіz danih epіzootichnih spalahіv sibіrki na terytorii' Ukrayi'ny (perіod 1994 – 2016 rr.) [Analysis of epizootic outbreaks of anthrax in Ukraine (1994 - 2016).]. Nauk. vіsnik vet. med: zbіrnik naukovih prac' [Scientifi c Bulletin of Veterinary Medicine: Collection of Scientifi c Papers]. Bila Tserkva, Issue 1 (127), no. 1 (127), pp. 87–95.

10. Skrypnyk, V.G., Koziy, R.V., Skrypnyk, A.V., Rublenko, I.O. (2014). Anthrax in dogs. Veterinarna medicina [Veterinary medicine]. no. 1 (215), pp. 14–17.

11. Instrukcii' z laboratornoi' diagnostyky sybirky u ljudej, v syrovyni tvarynnogo pohodzhennja ta ob’jektah dovkillja: Nakaz MOZ Ukrai'ny vid 21.08.2002, № 321 [Instructions for laboratory diagnostics of anthrax in humans, in raw materials of animal origin and environmental objects: Order of the Ministry of Health of Ukraine of 21.08.2002, No. 321.]. Available at: laws_2002.html.

12. Instrukcija 4.2.10–19–60–2005 [Instruction 4.2.10– 19–60–2005]. Laboratornaja diagnostika sibirskoj jazvy u ljudej i vydelenie vozbuditelja sibirskoj jazvy iz ob'ektov vneshnej sredy: Postanovlenie Glavnogo gosudarstvennogo sanitarnogo vracha Respubliki Belarus' ot 21 nojabrja 2005, № 181 [Laboratory diagnosis of anthrax in humans and the isolation of the causative agent of anthrax from environmental objects: Resolution of the Chief State Sanitary Doctor of the Republic of Belarus of November 21, 2005, No. 181].

13. Laboratornaja  diagnostika i obnaruzhenie vozbuditelja sibirskoj jazvy: metodicheskie ukazanija [Laboratory diagnosis and detection of anthrax pathogen: guidelines]. MUK 4.2.2413–08: Utverzhdeno Rukovoditelem federal'noj sluzhby po nadzoru v sfere zashhity prav potrebitelej i blagopoluchija cheloveka, Glavnym gosudarstvennym sanitarnym vrachom Rossijskoj Federacii G.G. Onishhenko ot 29 ijulja 2008 [MUK 4.2.2413–08: Approved by G.G., Head of the Federal Service for Supervision of Consumer Rights Protection and Human Well-Being, Chief State Sanitary Doctor of the Russian Federation Onishchenko on July 29, 2008]. 

14. Dragon, D.C., Rennie, R.P. (2001). Evalution of spore extraction and purifi cation methods for selective recovery of viable Bacillus anthracis spores. The Society for Applied Microbiology. no. 33, pp. 100–105.

15. Schuch, R., Fischetti, V.A. (2009). The secret life of the Anthrax agent Bacillus anthracis: bacteriophage mediated ecological adaptations. // Plos one. A Peer-Reviewed, one. Access journal. no. 4 (8). Available at:https://www.ncbi.nlm.

16. Zocenko, V.M., Rublenko, І.O., Andrіjchuk, A.V. (2018). Zagal'na mіkrobіologіja: metodichnі vkazіvki dlja praktichnoi' ta samostіjnoi' roboti studentіv fakul'tetu veterinarnoi' medicini z mіkrobіologіchnih metodіv doslіdzhen' [Mental practice: methodological instructions for practical and independent work of students in the Faculty of Veterinary Medicine with microbiological methods]. Bila Tserkva, 80 p.

17. Grimes, S.E. (2002). A Basic Laboratory Manual for the Small-Scale Production and Testing of I-2 Newcastle Disease Vaccine. Ten-fold serial dilutions. Appendix 5. no. 22, Availableat:

18. V.G. Skripnik, І.O. Rublenko, T.O. (2015). Laboratorna diagnostyka sybirky tvaryn, indykacija zbudnyka z patologichnogo ta biologichnogo materialu, syrovyny tvarynnogo pohodzhennja ta ob’jektiv navkolyshn'ogo seredovyshha: naukovo-metodychni rekomendacii' dlja zabezpechennja praktychnoi' ta samostijnoi' roboty fahivciv laboratorij ta naukovo-doslidnyh ustanov veterynarnoi' medycyny, vykladachiv ta studentiv fakul'tetiv veterynarnoi' medycyny VNZ [Laboratory diagnostics of animal anthrax, indication of pathogen and biological agent, raw material of animal origin and environmental objects: scientifi c and methodological recommendations for ensuring the practical and independent work of specialists of laboratories and research institutions of veterinary medicine, teachers and students]. Kyiv, 78 p.

PDF icon rublenko_2_2019.pdf2.46 MB