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MICROBIOLOGY COMMENT |
1 Laboratory of Food Microbiology and Immunology, Research Center for Animal Hygiene and Food Safety, Obihiro University of Agriculture and Veterinary Medicine, 2-11 Inada, Obihiro 080-8555, Hokkaido, Japan
2 Hokkaido Branch, National Institute of Animal Health, Sapporo, Hokkaido, Japan
Correspondence
Keiko Kawamoto
(kkeiko{at}obihiro.ac.jp)
We read with interest the comments made by Goossens et al. (2007)
concerning our recent publication (Enkhtuya et al., 2006). In our paper we demonstrated the specificity of rabbit anti-Bacillus anthracis (pXO1– and pXO2–) Pasteur II spore IgG and its protective effect in mice against lethal challenge by pXO1+ and pXO2+ B. anthracis spores. The following are our comments addressing their concerns.
1. We are well aware that Bacillus cereus and Bacillus thuringiensis are closely related to B. anthracis, as they are classified into one group based on genomic analysis; thus, we examined the cross-reactivity of our rabbit anti-anthrax spore IgG to these three species plus Bacillus subtilis by indirect immunofluorescence staining. Fluorescence images were all taken with the same exposure time and conditions in order to compare the relative fluorescence intensity. As clearly stated in the text, we obtained remarkably strong fluorescence from B. anthracis spores, while in contrast there was no detectable fluorescence from images of other tested Bacillus spores, including B. cereus, B. thuringiensis and B. subtilis, in addition to a negative control. Due to space constraints, these data were not shown in the original paper (Enkhtuya et al., 2006). However, we are happy to include these findings here (Fig. 1). We are certain that anti-spore IgG obtained by immunization using formalin-fixed spores of plasmidless Pasteur II strain reacts specifically with B. anthracis spores.
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; Stopa, 2000). The works cited by the authors as evidence against our work demonstrated cross-reactivity of fluorescein-conjugated anti-B. anthracis spore IgG among B. anthracis, B. cereus or B. thuringiensis by immunostaining (Phillips et al., 1983; Stopa, 2000). However, there are several differences between the methods used. For example, we used formalin-fixed spores of B. anthracis Pasteur II lacking pXO1 and pXO2 as antigens to immunize rabbits, whereas Phillips et al. immunized rabbits, with formalin-fixed spores of B. anthracis Vollum strain (Phillips et al., 1983), while mixtures of irradiated anthrax spores (Ames, Vollum and Sterne) were used in Stopa's work for immunization of goats (Stopa, 2000). Furthermore in order to determine fluorescence intensity, a quantitative immunofluorescence assay based on fibre optic microscopy was used in the work by Phillips et al. (1983), a flow cytometer was employed by Stopa (2000), while we used fluorescence microscopy to determine not only the intensity but also the location of fluorescence. Thus, the strain of spores, purity of immunogens and methods used in each study were different, and subsequently it is not surprising that the reactivity of antibodies is different in each study. In fact, as stated in their paper (Phillips et al., 1983), rabbit anti-Vollum IgG reacted well with both vegetative cells and spores of B. anthracis. However, our purified anti-spore IgG did react with endospores without cross-reacting with the vegetative form of B. anthracis as shown in Fig. 1
of our paper (Enkhtuya et al., 2006). Thus, the nature of the spore samples is likely to affect the reactivity of antibodies obtained. Previous studies have shown that high variation in fluorescence intensity can be obtained from spores of different Bacillus strains; although the antibody reacts with the three strains of B. anthracis (Ames, Vollum and Sterne), there are significant differences in the reactivity of antibody among them (Stopa, 2000). Similar findings have reported that various degrees of cross-reactivity are detected from various strains of B. cereus (Phillips et al., 1983). These findings suggest that molecular profiles expressed on the spore surface are variable, even among the same species. We have no doubt about the accuracy of the results published by other authors, but we are also confident about the results and conclusions demonstrated in our work.
2. (i) As clearly stated in our paper (Methods), the strain used in our study for anthrax challenge was Pasteur II strain carrying two virulent plasmids pXO1 and pXO2, which is atypical Pasteur II and produces toxins and capsule. The definition of ‘fully virulent’ is a contentious issue; however, one widely used definition involves B. anthracis being toxigenic and encapsulated (Koehler, 2000). Both plasmids are required for full pathogenicity, and this criterion is fulfilled by the strain used in our study. Indeed, all mice died as a result of infection with the Pasteur II containing pXO1 and pXO2 (5x103 spores per mouse intraperitoneally). Pasteur II is also recommended by the OIE (Office International des Epizooties, 2004) as a challenge strain to evaluate the efficacy and immunogenicity of Sterne 34F2 vaccines. Furthermore the ‘Carbosap’ strain mentioned by the authors (Goosens et al., 2007) as used as an attenuated vaccine strain in Italy is different from Pasteur II 74.12 strain as characterized by using a ninth genotype marker called pXO2-A (Muscillo et al., 2005). Carbosap vaccine strain also harbours pXO1 and pXO2, and is shown to be pathogenic in mice and guinea pigs (Fasanella et al., 2001). Pasteur-type vaccines, the first anthrax vaccine, varied greatly in their degree of attenuation. Some cultures were sufficiently virulent to kill the vaccinated animals, while others were too attenuated to give immunity (Ivins et al., 1986). Thus, it is no longer used as vaccine strain because of the heterogeneity. There is likely to be diversity within clones derived from original Pasteur strain.
(ii) The authors questioned the in vivo relevance of our protective humoral immune response experiments. We are working on active immunization experiments that support our published data, and hope to publish the data in the near future. We think anti-spore IgG performs its function at a very early stage of B. anthracis infection, as anthrax spores germinate rapidly into vegetative cells, and therefore pre-incubation of spores with IgG was performed based on this premise. Consistent with this, reduced protection was observed when anti-spore IgG was administered 30–40 min after spore inoculation, while no protection was observed by 24 h post-infection treatment (Fig. 6 in our paper, Enkhtuya et al., 2006). Thus, as stated in our paper, other virulence factors, such as protective antigen, are likely to be required in order to achieve full protection against the complex virulence of B. anthracis.
We acknowledge that intraperitoneal challenge of anthrax spores is not the natural route of infection, and that intranasal or intratracheal inoculation may better reflect human inhalation of anthrax. However, the infection model used in our study (intraperitoneal route of infection) has been widely employed by many investigators to study the pathogenesis of anthrax (Beedham et al., 2001; Perkins et al., 2005; Popov et al., 2004; Cote et al., 2006; Joyce et al., 2006). It is widely accepted that macrophages play a critical role in host–pathogen interactions of anthrax, irrespective of the mode of spore delivery. We agree with the opinion mentioned by Povov et al. (2004) that spores inoculated into the peritoneal cavity are transported to lymph nodes by resident macrophages by (or through) a mechanism similar to inhalation anthrax. Therefore, intraperitoneal delivery of B. anthracis spores appears to be an acceptable method for studying the pathogenicity of anthrax. No less important, due to consideration for the accuracy of spore delivery, safety concerns posed by the creation of spore-containing aerosols, and the requirement for expensive facilities and equipment, we chose intraperitoneal challenge for this study. Each mode of delivery has advantages and disadvantages; however, we are eager to examine various routes of infection in future studies.
3. (i, ii) As mentioned in a published report (Welkos et al., 2004), the classically used spore germination method of monitoring the decline of optical density is insensitive and inconsistent. Macrophages are required for B. anthracis spore germination and therefore we chose to examine the inhibitory effect of anti-spore IgG on germination in an in vitro macrophage system. We feel this system reflects the physiological requirements of spore germination. In our paper (Methods) it is clearly stated how the experiments were performed (in triplicate), including the total number of spores added. We do, however, apologize for not including numbers of total interacting spores, which were very similar between all samples. To calculate the number of total interacting spores, we enumerated c.f.u. of supernatants and washing fractions after 30 min incubation with spores (as non-interacting spores), and subtracted these values from the number added. Then, in order to differentiate from germinated spores, replicated vegetative cells and ungerminated spores, lysates were heat-treated and c.f.u. numbers were determined. Again we think the results adequately support our conclusions.
I hope we have addressed all your concerns surrounding our study and would like to point out that our study holds the promise of potentially useful antibodies. Certainly the editor and qualified reviewers of our manuscript thought the same and we hope that readers take into consideration that studies based on this antibody and plasmidless spores remain ongoing.
REFERENCES
Beedham, R. J., Turnbull, P. C. & Williamson, E. D. (2001). Passive transfer of protection against Bacillus anthracis infection in a murine model. Vaccine 19, 4409–4416.[CrossRef][Medline]
Cote, C. K., Van Rooijen, N. & Welkos, S. L. (2006). Roles of macrophages and neutrophils in the early host response to Bacillus anthracis spores in a mouse model of infection. Infect Immun 74, 469–480.
Enkhtuya, J., Kawamoto, K., Kobayashi, Y., Uchida, I., Rana, N. & Makino, S.-i. (2006). Significant passive protective effect against anthrax by antibody to Bacillus anthracis inactivated spores that lack two virulence plasmids. Microbiology 152, 3103–3110.
Fasanella, A., Losito, S., Trotta, T., Adone, R., Massa, S., Ciuchini, F. & Chiocco, D. (2001). Detection of anthrax vaccine virulence factors by polymerase chain reaction. Vaccine 19, 4214–4218.[CrossRef][Medline]
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Ivins, B. E., Ezzell, J. W., Jr, Jemski, J., Hedlund, K. W., Ristroph, J. D. & Leppla, S. H. (1986). Immunization studies with attenuated strains of Bacillus anthracis. Infect Immun 52, 454–458.
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Koehler, T. M. (2000). Bacillus anthracis. In Gram-Positive Pathogens, pp. 520. Edited by V. A. Fischetti, R. P. Novik, J. J. Ferretti, D. A. Portnoy & J. I. Rood. Washington, DC: American Society for Microbiology.
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.
Office International des Epizooties (2004). Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Paris: Office International des Epizooties.
Perkins, S. D., Flick-Smith, H. C., Garmory, H. S., Essex-Lopresti, A. E., Stevenson, F. K. & Phillpotts, R. J. (2005). Evaluation of the VP22 protein for enhancement of a DNA vaccine against anthrax. Genet Vaccines Ther 3, 3.[CrossRef][Medline]
Phillips, 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.
Popov, S. G., Popova, T. G., Grene, E., Klotz, F., Cardwell, J., Bradburne, C., Jama, Y., Maland, M., Wells, J. & other authors (2004). Systemic cytokine response in murine anthrax. Cell Microbiol 6, 225–233.[CrossRef][Medline]
Stopa, P. J. (2000). The flow cytometry of Bacillus anthracis spores revisited. Cytometry 41, 237–244.[Medline]
Welkos, S. L., Cote, C. K., Rea, K. M. & Gibbs, P. H. (2004). A microtiter fluorometric assay to detect the germination of Bacillus anthracis spores and the germination inhibitory effects of antibodies. J Microbiol Methods 56, 253–265.[CrossRef][Medline]
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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