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Institute of Bacterial Infections and Zoonoses (IBIZ)

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National Reference Laboratory for Anthrax

Anthrax is a contagious and potentially fatal disease caused by the bacterium Bacillus anthracis. It is a Gram-positive, facultative anaerobic, spore-forming, and rod-shaped bacterium that primarily affects herbivores, such as cattle, sheep, goats, horses, but may infect also other farm animals and wildlife as well. In addition to a capsule, virulent strains possess a plasmid-encoded protein toxin, which consists of a protective, a lethal and an edema factor. Spores may stay viable for decades within the soil environment and pose persistent risk for new outbreaks. Animals primarily contract the disease through ingestion of spores via feed or inhalation. Grazing is the most common mode of uptake of spores, while insects may also transmit the bacterium between animals. Carnivores get infected when feeding on cadavers of fallen animals. The incubation period in infected animals ranges from one to fourteen days, and the characteristic signs of the disease are sudden death, edematous swelling along the neck, and bloody exudate from all orifices.

Humans may contract anthrax when in contact with infected animals or contaminated animal products, such as meat, bones, blood, wool or hair, and hides. B. anthracis spores may be ingested or inhaled or enter the body through skin abrasions and wounds. Depending on the pathway of entry, three forms of anthrax can develop: cutaneous, pulmonary, or intestinal anthrax. 

Anthrax occurs worldwide and is currently more prevalent in warmer climate zones such as Southeastern Europe, South America, Africa, and Southeast Asia. The occurrence of the disease is often also associated with natural disasters such as flooding, heavy rain, landslides, or excavations. In Europe, anthrax incidence is low, and cases occur only occasionally and locally. Between 2005 and 2022, a total of 267 cases of anthrax were registered at the World Organization of Animal Health (WOAH). 251 cases were seen in domestic animals and 16 in wildlife. In Germany, anthrax occurs sporadically, since 2009 only 5 cases have been reported nationwide.

Pathogen detection involves cultivation and subsequent molecular detection of virulence plasmid borne toxin genes. 

The main tasks of the National Reference Laboratory are support the federal state laboratories by conducting confirmation tests and providing regular laboratory comparison tests.

Anthrax is a disease that may be associated with severe economic losses in animal production and human infections. About 1.8 billion people and their livestock live in anthrax-suitable areas. Disastrous outbreaks are seen in wildlife threatening also endangered species like African elephants and lions. Anthrax also proved to be a notorious agent of bioterrorism and subject of biological weapon programs of various states. Spores are markedly resistant to heat, cold, pH, drying, chemicals and irradiation and environments may stay contaminated for undefined time. The disease is spread worldwide but is most common in Asia (Middle and Far East, India), Africa, and South America. In Europe, the Mediterranean region and Eastern Europe are most affected. In Germany there are many regions especially, areas where anthrax infected animals were disposed or tanneries were placed may still be contaminated, but anthrax in animals has only been detected sporadically. From 1981 to 2002, 41 anthrax outbreaks in animals (new outbreaks, farmsteads) were reported.

  • training of and support for the diagnostic laboratories of the German federal states,
  • clarification of unclear findings,
  • development of new diagnostic methods,
  • providing regular laboratory comparison tests,
  • provision of reference materials.

The laboratory set up of the NRL for anthrax in Jena is designed to quickly confirm or rule out whether a sample is contaminated with Bacillus anthracis or not. Our laboratory is prepared to receive samples from outbreaks or alleged use. Isolating the bacteria can be challenging in old or decomposed animal samples, processed products from animals that have died of anthrax, or environmental samples due to the presence of other bacteria. Thus, one of the tasks of the National Reference Laboratory is to optimize diagnostic procedures for complex samples obtained from tissues, organs, powder, blood, and farm environmental samples.

Due to morphological similarities, the differentiation of Bacillus species is of particular practical importance. Thus, specific testing methods are applied e.g. cultivation on anthrax blood agar and chromogenic culture media. B. anthracis colonies appear as pinhead-sized and grow within 4-5 hours on the chromogenic culture media. After 18-20 hours, B. anthracis forms large, white colonies without hemolysis, while other species show different characteristics. To confirm the identity of the isolated cultures or suspicious colonies, plasmid detection using PCRs is conducted. WGS and subsequent use of bioinformatic analysis will help to do risk assessment and to analyse strains for tracing.

Handling of materials potentially containing B. anthracis requires authorization and has to be carried out in a safety level 3 laboratory.

  • advice on diagnostics, including material transport, culture media, cultivation methods, and risk assessment,
  • characterization of bacterial isolates using phenotypic methods and up to date molecular analysis and bioinformatics,
  • development and optimization of molecular means for pathogen detection, including nested and real-time PCR,
  • maintenance of a strain collection of genetically typed isolates,

  • recording and analysis of anthrax outbreaks in Germany for epidemiological purposes.

    For diagnostic requirements, please contact the NRL in advance!
     

Literature

Beyer W, Glöckner P, Otto J, Böhm R (1995) A nested PCR method for the detection of Bacillus anthracis in environmental samples collected from former tannery sites. Microbiol Res 150, 179-186. doi. 10.1016/S0944-5013(11)80054-6 PubMed

Fasanella A, Di Taranto P, Garofolo G, Colao V, Marino L, Buonavoglia D, Pedarra C, Adone R, Hugh-Jones M (2013) Ground Anthrax Bacillus Refined Isolation (GABRI) method for analyzing environmental samples with low levels of Bacillusanthracis contamination. BMC Microbiol 13, 167. doi: 10.1186/1471-2180-13-167 PubMed

Kozytska T, Bassiouny M, Chechet O, Ordynska D, Galante D, Neubauer H, Wareth G (2023) Retrospective analysis of official data on Anthrax in Europe with a special reference to Ukraine. Microorg 11(5), 1294. doi: 10.3390/microorganisms 11051294 PubMed

Peng H, Ford V, Frampton EW, Restaino L, Shelef LA, Spitz H (2001) Isolation and enumeration of Bacillus cereus from foods on a novel chromogenic plating medium. Food Microbiol 18, 231-238. doi.10.1006/fmic.2000.0369. ScienceDirect

World Organization for Animal Health (WOAH): Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. CHAPTER 3.1.1. ANTHRAX (PDF)