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MICROBIOLOGY COMMENT |
1 Unité Toxines et Pathogénie Bactérienne, Institut Pasteur, 28 Rue du Dr Roux, 75724 Paris Cedex 15, France
2 URA 2172, CNRS, 28 Rue du Dr Roux, 75724 Paris Cedex 15, France
Correspondence
Michèle Mock
(mmock{at}pasteur.fr)
Passive protection mediated by antibodies directed against Bacillus anthracis inactivated spores has been suggested by Enkhtuya et al. (2006)
in a recent paper in this journal. In our view, the main conclusions drawn in this article are overstatements in regard to the data presented and to the currently published studies, and thus, are misleading.
1. The authors report as ‘data not shown’ that rabbit IgG directed against anti-formaldehyde inactivated (FIS) B. anthracis spores do not cross-react with spores of Bacillus cereus and Bacillus thuringiensis. This is surprising as spore surface components of B. anthracis, B. cereus and B. thuringiensis share common epitopes. In the course of studies on spore surface structures, we and others have repeatedly encountered difficulties in obtaining B. anthracis spore-specific antibodies; a high level of cross-reactivity is consistently observed with most strains of B. cereus (for example Philips et al., 1983; Stopa, 2000; Sylvestre, 2003). This point is well known to those working on specific detection of spores of B. anthracis relative to B. cereus and B. thuringiensis. Indeed this is why the design of a means for rapid and specific detection of B. anthracis spores is still one of the aims of current bioterrorism countermeasure programmes.
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, is not ‘fully virulent’ as stated in the article by Enkhtuya et al. (2006). Moreover it has been used until recently as a live veterinary vaccine in Italy (World Health Organization, 1998). It is known that some attenuated strains still possess both virulence plasmids and are not considered fully virulent strains (Cataldi et al., 2000; Muscillo et al., 2005). Mislabelling of the challenge strain used thus compromises the significance of the protection data reported. (ii) The experimental conditions used in this study are very far from the natural in vivo conditions in which a protective humoral immune response functions. Ideally, passively transferred humoral immunity should mimic what would be observed in in vivo immunized hosts in which circulating antibodies could be recruited and exert their protective function in the infected sites. Protection should thus be observed even if the B. anthracis spore challenge is performed in a different location than the administered antibodies (for example challenge by subcutaneous route and antibody injection by intraperitoneal or intravenous route). The experimental procedures reported by Enkhtuya et al. (2006) artificially favour spore–antibody interaction: spores are either coated and opsonized with antibodies before infection, giving an 80 % survival, or injected in the same body compartment as the antibodies (the peritoneum) with a short delay of 30–40 min between each injection, leading to only 40 % protection. It would be informative about the in vivo relevance of this passive transfer of protection (a) to follow the level of protection achieved when more time is allowed to elapse between spore challenge and antibody inoculation, and (b) to ascertain the level of protection achieved when the challenge is performed by the classical subcutaneous route. Indeed, in our hands, in experiments of homologous passive transfer of FIS-immune serum (mouse FIS-immune serum intraperitoneally transferred in recipient mice challenged subcutaneously) we did not observe any protective effects (Glomski et al., 2007).
3. The cell infection experiments, from which the inhibition of germination by the anti-FIS IgG is concluded, lack essential information. (i) The informative experiment in this regard would have been to follow the optical density decrease of a pure spore suspension as classically used by many colleagues studying germination kinetics and as already described for B. anthracis (Titball & Manchee, 1987; Moir, 1990; Welkos et al., 2001). (ii) In a similar cell infection experimental system, Welkos et al. (2002) have shown that phagocytosis by macrophages, and subsequent germination and killing of the spores, was enhanced by antibody coating of the spores. Since, in the study by Enkhtuya et al. (2006), all germinated spores are heat-killed before c.f.u. counting is performed, essential information about total and germinated spore c.f.u. counts is lacking. This hampers evaluation of the actual effect of antibody coating on the extent of germination, and thus does not adequately support the conclusions drawn.
In summary, the results reported by Enkhtuya et al. (2006) do not appear relevant, as such, to an in vivo protective role of anti-spore antibodies in an actively immunized animal. However, the study, which relies on an experimentally forced system, confirms the ability of rabbit antibodies reactive with B. anthracis antigens to stimulate mouse macrophages to efficiently kill the bacteria and thus help control infection (Welkos et al., 2001, 2002).
REFERENCES
Cataldi, A., Mock, M. & Bentancor, L. (2000). Characterization of Bacillus anthracis strains used for vaccination. J Appl Microbiol 88, 648–654.[CrossRef][Medline]
Enkhtuya, J., Kawamoto, K., Kobayashi, Y., Uchida, I., Rana, N. & Makino, S.-I. (2006). Significant passive protective effects against anthrax by antibody to Bacillus anthracis inactivated spores that lack two virulence plasmids. Microbiology 152, 3103–3110.
Glomski, I. J., Corre, J.-P., Mock, M. & Goossens, P. L. (2007). IFN-
producing CD4 T lymphocytes mediate spore-induced immunity to capsulated Bacillus anthracis. J Immunol (in press).
Moir, A. (1990). The genetics of bacterial spore germination. Annu Rev Microbiol 44, 531–553.[CrossRef][Medline]
Muscillo, M., La Rosa, G., Sali, M., De Carolis, E., Adone, R., Ciuchini, F. & Fasanella, A. (2005). Validation of a pXO2-A PCR assay to explore diversity among Italian isolates of Bacillus anthracis strains closely related to the live attenuated Carbosap vaccine. J Clin Microbiol 43, 4758–4765.
Philips, A. P., Martin, K. L. & Broster, M. G. (1983). Differentiation between spores of Bacillus anthracis and Bacillus cereus by a quantitative immunofluorescence technique. J Clin Microbiol 17, 41–47.
Stopa, P. J. (2000). The flow cytometry of Bacillus anthracis spores revisited. Cytometry 41, 237–244.[Medline]
Sylvestre, P. (2003). Structural and functional analysis of the Bacillus anthracis exosporium. PhD thesis, Université Paris VII, France.
Titball, R. W. & Manchee, R. J. (1987). Factors affecting the germination of spores of Bacillus anthracis. J Appl Microbiol 62, 269–273.[Medline]
Uchida, I., Sekizaki, T., Hashimoto, K. & Terakado, N. (1985). Association of the encapsulation of Bacillus anthracis with a 60 megadalton plasmid. J Gen Microbiol 131, 363–367.[Medline]
Welkos, S., Little, S., Friedlander, A., Fritz, D. & Fellows, P. (2001). The role of antibodies to Bacillus anthracis and anthrax toxin components in inhibiting the early stages of infection by anthrax spores. Microbiology 147, 1677–1685.
Welkos, S., Friedlander, A., Weeks, S., Little, S. & Mendelson, I. (2002). In vitro characterisation of the phagocytosis and fate of anthrax spores in macrophages and the effects of anti-PA antibody. J Gen Microbiol 51, 821–831.
World Health Organization (1998). Guidelines for the Surveillance and Control of Anthrax in Humans and Animals. WHO/EMC/ZDI/98/6, 3rd edn, p. 80. Geneva: World Health Organization.
This article has been cited by other articles:
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  C. K. Cote, J. Bozue, K. L. Moody, T. L. DiMezzo, C. E. Chapman, and S. L. Welkos Analysis of a novel spore antigen in Bacillus anthracis that contributes to spore opsonization Microbiology, February 1, 2008; 154(2): 619 - 632. [Abstract] [Full Text] [PDF]
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J. Enkhtuya, S.-i. Makino, I. Uchida, and K. Kawamoto In reply Microbiology, February 1, 2007; 153(2): 302 - 304. [Full Text] [PDF]
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