Molecular characterization of Bacillus anthracis isolates recovered from nomic and nonnomic hosts

Anthrax, which is primarily a disease of herbivores, is known to infect many animal species. In order to break the cycle of anthrax infection, it is important to identify the potential hosts or reservoirs of its causative agent. Therefore, this study aimed to characterize Bacillus anthracis isolates recovered from a variety of hosts and environments during anthrax sporadic in the Kars and Kayseri regions of Turkey. For this purpose, a total of 23 B. anthracis field isolates obtained from human, cattle, sheep, dog, horse, puma, soil, and fodder were used. In addition, the Sterne vaccine strain was included. All B. anthracis isolates were confirmed via molecular tests using plasmid-based PCR and 16S rRNA gene sequencing. All were found to be positive for protective antigen (PA) and capsule (Cap) genes. The presence of two specific 16S rRNA positions (1139 and 1148) with nucleotide mismatches was used to differentiate between B. anthracis species in the Bacillus cereus group, which has a relatively high (>99%) 16S rRNA sequence similarity. All of our isolates, including the Sterne vaccine strain, were classified in the same 16S rRNA type (Type 6) that has been reported previously as being the predominant type for B. anthracis isolates.

PDF

___

1. World Health Organization (WHO). Anthrax in Humans and Animals. 4th ed. WHO, Geneva, Switzerland, 2008. Pp. 1-208.
2. Hugh-Jones ME, de Vos V. Anthrax and wildlife. In: Bengis RG (editor). Infectious Diseases of Wildlife: Detection, Diagnosis and Management (Part Two). Revue Scientifique et Technique (International Office of Epizootics) 2002. Pp. 359-383.
3. Ekebas G, Atasever A, Gram DY, Karakaya E, Abay S et al. A case of Anthrax in two captive pumas (Puma concolor). The Journal of Veterinary Medical Science 2018; 80: 1875-1880.
4. Doganay M, Demiraslan H. Human anthrax as a re-emerging disease. Recent Patents on Anti-Infective Drug Discovery 2015; 10: 10-29.
5. Buyuk F, Sahin M, Cooper C, Celebi O, Saglam AG et al. The effect of prolonged storage on the virulence of isolates of Bacillus anthracis obtained from environmental and animal sources in the Kars Region of Turkey. FEMS Microbiology Letters 2015; 362: fnv102.
6. Beyer W, Glockner P, Otto J, Böhm R. A nested PCR method for the detection of Bacillus anthracis in environmental samples collected from former tannery sites. Microbiology Research 1995; 150: 179-186.
7. Sacchi CT, Whitney AM, Mayer LW, Morey R, Steigerwalt A et al. Sequencing of 16S rRNA gene: A rapid tool for identification of Bacillus anthracis. Emerging Infectious Diseases 2002; 8: 1117-1123.
8. Bavykin SG, Lysov YP, Zakhariev V, Kelly JJ, Jackman J et al. Use of 16S rRNA, 23S rRNA, and gyrB gene sequence analysis to determine phylogenetic relationships of Bacillus cereus group microorganisms. Journal of Clinical Microbiology 2004; 42; 3711-3730.
9. Roonie A, Majumder S, Kingston JJ, Parida M. Molecular characterization of B. anthracis isolates from the anthrax outbreak among cattle in Karnataka, India. BMC Microbiology 2020; 20: 1-12.
10. Lane DJ. 16S/23S rRNA Sequencing. In: Stackebrandt E and Goodfellow M (editors). Nucleic Acid Techniques in Bacterial Systematic. John Wiley and Sons, New York, 1991. Pp. 115-175.
11. Felsenstein J. Evolutionary trees from DNA sequences: A maximum likelihood approach. The Journal of Molecular Evolution 1981; 17: 368-376.
12. Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution (N. Y) 1985; 39: 783-791.
13. Nishimaki T, Sato K. An Extension of the Kimura twoparameter model to the natural evolutionary process. The Journal of Molecular Evolution 2019; 87: 60-67.
14. Hoffmaster AR, Koehler TM. Control of virulence gene expression in Bacillus anthracis. Journal of Applied Microbiology 1999; 87: 279-281.
15. Anonymous. Handbook on Contagious and Infectious Diseases in Animals. Government of India Press, Delhi, India, 1945.
16. Schuch R, Fischetti VA. The secret life of the anthrax agent Bacillus anthracis: Bacteriophage-mediated ecological adaptations. PLoS One 2009; 4: e6532.
17. Sterne M. Anthrax. In: Stableforth AW and Galloway IA (editors). Infectious Diseases of Animals. Vol. 1. Diseases Due to Bacteria. Butterworths, London, UK, 1959. Pp. 16-52.
18. Gajardo G, Uberti B, Paredes E. Anthrax in a horse and postexposure interventions. Journal of Equine Veterinary Science 2016; 39: S37-S38.
19. Durmaz R, Doganay M, Sahin M, Percin D, Karahocagil MK et al. Molecular epidemiology of the Bacillus anthracis isolates collected throughout Turkey from 1983 to 2011. The European Journal of Clinical Microbiology & Infectious Diseases 2012; 31: 2783-2790.
20. Khmaladze E, Su W, Zghenti E, Buyuk F, Sahin M et al. Molecular genotyping of Bacillus anthracis strains from Georgia and Northeastern Part of Turkey. Journal of Bacteriology & Mycology 2017; 4: 1053.
21. Topluoglu S, Aktas D, Celebi B, Kara F, Doganay M et al. Human anthrax in Turkey: A ten years’ experience (2009- 2018). Tropical Doctor 2021; 51: 80-83.
22. Blackburn JK, Skrypnyk A, Bagamian KH, Nikolich MP, Bezymennyi M et al. Anthrax in a backyard domestic dog in Ukraine: A case report. Vector Borne Zoonotic Disease 2014; 14: 615-617.
23. Langston C. Postexposure management and treatment of anthrax in dogs-executive councils of the American Academy of Veterinary Pharmacology and Therapeutics and the American College of Veterinary Clinical Pharmacology. The American Association of Pharmaceutical Scientists 2005; 7: E272-E273.
24. Bauer JW, Logan KA, Sweanor LL, Boyce WM. Scavenging behavior in puma. The Southwestern Naturalist 2005; 50: 466- 471.
25. Turnbull PC, Doganay M, Lindeque PM, Aygen B, McLaughlin J. Serology and anthrax in humans, livestock and Etosha National Park wildlife. Epidemiology and Infection 1992; 108: 299-313.
26. Somers F. Anthrax amongst wild animals in captivity. Veterinary Record 1911; 23: 543-544.
27. Van Ness GB. Ecology of anthrax. Science 1971; 172: 1303- 1307.
28. Buyuk F, Celebi O, Linley E, Cooper C, Doganay M et al. Does environmental exposure to Bacillus anthracis spores lead to sub-clinical infection? In: The International Conference on Bacillus anthracis, B. cereus, and B. thuringiensis; Canadian; 2013.
29. Chakravorty S, Helb D, Burday M, Connell N, Alland D. A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria. The Journal of Microbiological Methods 2007; 69: 330-339.
30. Hakovirta JR, Prezioso S, Hodge D, Pillai SP, Weigel LM. Identification and analysis of informative single nucleotide polymorphisms in 16S rRNA gene sequences of the Bacillus cereus group. Journal of Clinical Microbiology 2016; 54: 2749- 2756.
31. Alexander KA, Carlson CJ, Lewis BL, Getz WM, Marathe MV et al. The ecology of pathogen spillover and disease emergence at the human-wildlife-environment interface. The Connections Between Ecology and Infectious Disease 2018; 267-298.