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Journal of Clinical Microbiology, September 2005, p. 4758-4765, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4758-4765.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

M. Muscillo,^1* G. La Rosa,¹ M. Sali,¹ E. De Carolis,¹ R. Adone,² F. Ciuchini,² and A. Fasanella³

Department of Environment and Primary Prevention,¹ Department of Food and Veterinary Medicine, Istituto Superiore Sanità Roma, Rome, Italy,² Department of Anthrax Reference Institute of Italy at Istituto Zooprofilattico Sperimentale di Puglia e Basilicata, Foggia, Italy³

Received 10 February 2004/ Returned for modification 21 September 2004/ Accepted 4 May 2005

Several circulating Bacillus anthracis strains isolated in Italy and belonging to the A1.a cluster, genotype 3 (A1.a-3) are genotypically indistinguishable from Carbosap, a live attenuated vaccine strain, containing both pXO1 and pXO2 plasmids. The genotype was assessed by using eight-locus multilocus variable-number tandem repeat analysis. We describe here the use of a ninth locus able to explore variability among strains that have the same genotype. It is important to be able to genotype the wild isolate of B. anthracis strains from outbreaks of anthrax in areas where Carbosap vaccination of cattle and sheep is common practice. A total of 27 representative field strains isolated in Italy and four vaccinal strains, namely, Carbosap, Sterne, Pasteur I, and Pasteur II, were characterized by a ninth marker, called pXO2-A. Twenty-three field strains were genotype 3 and therefore identical to Carbosap. The marker was in the pXO2 plasmid and is based on the polymorphism of the already-known VX2-3 locus. Detection was obtained by PCR with fluorescence-labeled forward primers in order to produce appropriate fragments for capillary electrophoresis with an ABI 310 genetic analyzer. Genetic relationships showed heterogeneity in all of the examined samples. Interestingly, with respect to genotype 3, samples grouped into eight different subtypes, A to H, and the subtype G, had only two samples indistinguishable from Carbosap. The results of the present study confirm the validity of a hierarchical progressive protocol for discrimination among closely related isolates.

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* Corresponding author. Mailing address: Dipartimento Ambiente e Connessa Prevenzione Primaria, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Phone: 39 06 49902718. Fax: 39 06 49902718. E-mail: muscillo{at}iss.it.

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Journal of Clinical Microbiology, September 2005, p. 4758-4765, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4758-4765.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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