Ground Anthrax Bacillus Refined Isolation (GABRI) method for analyzing environmental samples with low levels of Bacillus anthracis contamination

BMC Microbiology

Martin Hugh-Jones⁴  Rosanna Adone³  Carmine Pedarra¹  Domenico Buonavoglia²  Leonardo Marino¹  Valeriana Colao²  Giuliano Garofolo⁵  Pietro Di Taranto¹  Antonio Fasanella¹ 

[1] Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Anthrax Reference Institute of Italy, Foggia, Italy;Departement of Veterinary Medicine, University of Bari, Bari, Italy;Istituto Superiore di Sanità, Roma, Italy;Department of Environmental Sciences School of the Coast & Environment, Louisiana State University, Baton Rouge, LA 70803-5703, USA;Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Teramo, Italy

关键词: Method;    Isolation;    Contaminated soil;    Bacillus anthracis;   

Others  :  1143417
DOI  :  10.1186/1471-2180-13-167

 received in 2013-04-04, accepted in 2013-07-03,  发布年份 2013

【 摘 要 】

Background

In this work are reported the results of a qualitative analytical method capable of detecting Bacillus anthracis spores when they are present in very low concentration in the soil. The Ground Anthrax Bacillus Refined Isolation (GABRI) method, assessed in our laboratory, was compared with the classic method. The comparison involved artificially anthrax-contaminated soil samples (500 spores/7.5 grams soil) and naturally contaminated soil samples collected in Bangladesh during a field investigation.

Results

The results indicated that, in contrast to the classic method, the GABRI method was able to detect B.anthracis in all contaminated samples. The GABRI method produces a more sensitive measure of anthrax spore presence significantly different from the standard method. In particular, the latter is more sensitive to the presence of normal soil contaminants.

Conclusion

The main feature of the GABRI method is its ability to strongly reduce the presence of the environmental contaminants, which being much more numerous than B. anthracis tend to inhibit their germination and growth making it extremely difficult to visualize any colonies. The reduction of the microbial environment also allows one to be able to culture and test a larger quantity of potentially contaminated soil and to isolate B. anthracis when the spores are present in very low concentrations in the soil.

【 授权许可】

   
2013 Fasanella et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

【 参考文献 】

• [1]WHO: Integrated control of neglected zoonotic diseases in Africa: applying the ‘One healt Concept’. Geneva: WHO Document Production Services; 2009.
• [2]Smith KL, DeVos V, Bryden H, Price LB, Hugh-Jones ME, Keim P: Bacillus anthracis diversity in Kruger National Park. J Clin Microbiol 2000, 38(10):3780-3784.
• [3]Higgins CH: Anthrax. In Health of Animals Branch, Bulletin 23. Ottawa: Department of Agriculture; 1916:3-8.
• [4]Minett FC, Dhanda MR: Multiplication of Bacillus anthracis and Cl. Chauvoei in soil and water. Indian journal of veterinary science and animal husbandry 1941, 11:308-321.
• [5]Van Ness G, Stein CD: Soils of the United States favorable for anthrax. J Am Vet Med Assoc 1956, 128(1):7-12.
• [6]Van Ness GB: Ecology of anthrax. Science 1971, 172(3990):1303-1307.
• [7]Hugh-Jones M, Blackburn J: The ecology of Bacillus anthracis. Molecular aspect of medicine 2009, 30(6):356-367.
• [8]Turnbull PC: Definitive identification of Bacillus anthracis–a review. J Appl Microbiol 1999, 87(2):237-240.
• [9]Dragon DC, Rennie RP: Evaluation of spore extraction and purification methods for selective recovery of viable Bacillus anthracis spores. Lett Appl Microbiol 2001, 33:100-105.
• [10]Marston CK, Beesley C, Helsel L, Hoffmaster AR: Evaluation of two selective media for the isolation of Bacillus anthracis. Lett Appl Microbiol 2008, 47(1):25-30.
• [11]Gulledge JS, Luna VA, Luna AJ, Zartman R, Cannons AC: Detection of low numbers of Bacillus anthracis spores in three soils using five commercial DNA extraction methods with and without an enrichment step. J Appl Microbiol 2010, 109(5):1509-1520.
• [12]Ryu C, Lee K, Yoo C, Seong WK, Oh HB: Sensitive and rapid quantitative detection of anthrax spores isolated from soil samples by real-time PCR. Microbiol Immunol 2003, 47(10):693-699.
• [13]Fasanella A, Garofolo G, Hossain MJ, Shamsuddin M, Blackburn JK, Hugh-Jones M: Bangladesh anthrax outbreaks are probably caused by contaminated livestock feed. Epidemiol Infect 2012, 20:1-8.
• [14]Fasanella A, Scasciamacchia S, Garofolo G: The behaviour of virulent Bacillus anthracis strain AO843 in rabbits. Vet Microbiol 2009, 133:208-209.
• [15]Office International des Epizooties: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 5th edition. Paris, France: OIE;
• [16]Turnbull PC, Frawley DA, Bull RL: Heat activation/shock temperatures for Bacillus anthracis spores and the issue of spore plate counts versus true numbers of spores. J Microbiol Methods 2007, 68(2):353-357.
• [17]Fasanella A, Losito S, Trotta T, Adone R, Massa S, Ciuchini F, Chiocco D: Detection of anthrax vaccine virulence factors by polymerase chain reaction. Vaccine 2001, 19(30):4214-4218.
• [18]Bland JM, Altman DG: Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986, 327(8476):307-310.
• [19]Rönner U, Husmark U, Henriksson A: Adhesion of bacillus spores in relation to hydrophobicity. J Appl Bacteriol 1990, 69(4):550-556.
• [20]Schuch R, Fischetti VA: The secret life of the anthrax agent bacillus anthracis: bacteriophage-mediated ecological adaptations. PLoS One 2009, 4(8):e6532.