ORIGINAL ARTICLE Analysis of a newly discovered antigen of Bacillus cereus biovar anthracis for its suitability in specific serological antibody testing S. Dupke 1 , A. Barduhn 1 , T. Franz 1 , F.H. Leendertz 2 , E. Couacy-Hymann 3 , R. Grunow 1 and S.R. Klee 1 1 Robert Koch-Institute, Centre for Biological Threats and Special Pathogens (ZBS2), Berlin, Germany 2 Robert Koch-Institute, Epidemiology of Highly Pathogenic Microorganisms (P3), Berlin, Germany 3 Laboratoire National d’Appui au Developpement Agricole (LANADA), Laboratoire Central Veterinaire de Bingerville (LCVB), Bingerville, C^ ote d’Ivoire Keywords Bacillus anthracis, Bacillus cereus biovar anthracis, immune response, protective antigen, pXO2-60, recombinant protein, serological assay. Correspondence Silke R. Klee, Robert Koch-Institute, Centre for Biological Threats and Special Pathogens (ZBS2), Seestr. 10, 13353 Berlin, Germany. E-mail: klees@rki.de 2018/1109: received 2 March 2018, revised 31 August 2018 and accepted 12 September 2018 doi:10.1111/jam.14114 Abstract Aims: The aim of this work was to identify a protein which can be used for specific detection of antibodies against Bacillus cereus biovar anthracis (Bcbva), an anthrax-causing pathogen that so far has been described in African rainforest areas. Methods and Results: Culture supernatants of Bcbva and classic Bacillus anthracis (Ba) were analysed by gel electrophoresis, and a 35-kDa protein secreted only by Bcbva and not Ba was detected. The protein was identified as pXO2-60 by mass spectrometry. Sequence analysis showed that Ba is unable to secrete this protein due to a premature stop codon in the sequence for the signal peptide. Immunization of five outbred mice with sterile bacterial culture supernatants of Bcbva revealed an immune response in ELISA against pXO2-60 (three mice positive, one borderline) and the protective antigen (PA; four mice). When supernatants of classic Ba were injected into mice or human sera from anthrax patients were analysed, only antibodies against PA were detected. Conclusions: In combination with PA, the pXO2-60 protein can be used for the detection of antibodies specific against Bcbva and discriminating from Ba. Significance and Impact of the Study: After further validation, serological assays based on pXO2-60 can be used to perform seroprevalence studies to determine the epidemiology of B. cereus bv anthracis in affected countries and assess its impact on the human population. Introduction Anthrax is a globally distributed zoonotic disease caused by the spore-forming bacterium Bacillus anthra- cis. Especially in developing countries, it causes consid- erable livestock and wildlife losses (WHO 2008). Humans most commonly contract anthrax from han- dling infected animals or contaminated animal products (Beyer and Turnbull 2009). Depending on the portal of entry, cutaneous, gastrointestinal or inhalational forms of anthrax can develop (WHO 2008). Severe infections after injection of contaminated drugs have also been reported (Hicks et al. 2012; Grunow et al. 2013). The high pathogenicity of B. anthracis is primarily caused by the anthrax toxins and the poly-c-D-glutamic acid capsule (Mock and Fouet 2001). The toxins comprise three proteins, protective antigen (PA), lethal factor (LF) and oedema factor (Moayeri et al. 2015). The toxin and capsule synthesis genes are located on the two virulence plasmids pXO1 and pXO2, respec- tively (Okinaka et al. 1999). Expression of these genes is induced by growth in CO 2 atmosphere and con- trolled by the global regulator AtxA (Mock and Mignot 2003). Journal of Applied Microbiology 126, 311--323 © 2018 The Society for Applied Microbiology 311 Journal of Applied Microbiology ISSN 1364-5072