Bacillus anthracis Virulence in Guinea Pigs Vaccinated with Anthrax Vaccine Adsorbed Is Linked to Plasmid Quantities and Clonality

Abstract

Bacillus anthracis is a bacterial pathogen of great importance, both historically and in the present. This study presents data collected from several investigations and indicates that B. anthracis virulence is associated with the clonality and virulence of plasmids pXO1 and pXO2. Guinea pigs vaccinated with Anthrax Vaccine Adsorbed were challenged with 20 B. anthracis isolates representative of worldwide genetic diversity. These same isolates were characterized with respect to plasmid copy number by using a novel method of quantitative PCR developed for rapid and efficient detection of B. anthracis from environmental samples. We found that the copy numbers for both pXO1 and pXO2 differed from those in previously published reports. By combining the data on survival, plasmid copy numbers, and clonality, we developed a model predicting virulence. This model was validated by using a randomly chosen set of 12 additional B. anthracis isolates. Results from this study will be helpful in future efforts to elucidate the basis for variation in the virulence of this important pathogen.

In silico and in vitro evaluation of PCR-based assays for the detection ofBacillus anthracischromosomal signature sequences

“…The results obtained using the S-layer, 40 BA813, 35 and SASP 44 assays displayed a lower agreement among laboratories (k values of 0.5-0.8). In general, the three methods had relative low diagnostic sensitivity and specificity compared with the BA5345, PL3, and BA5357 assays, indicating that these methods have a lower performance both in detecting B. anthracis in truly contaminated samples and in declaring truly non-contaminated samples as free of B. anthracis.…”

“…30 These genetic markers provide limited specificity and require additional timeconsuming and labor-intensive post-PCR analysis steps. Other areas of the chromosome have also been investigated as potential DNA-targets for identification purposes, including the so-called BA813 [31][32][33][34][35][36][37][38] and BA5510 sequences, 19 genes bclB, 39 sap, 40,41 saspB, 5,42 and sspE, 22,43 the B-type small acid-soluble spore protein gene (SASP), 44 a glycosyltransferase group 1 family protein, 45 a protein showing similarities with an abhydrolase, 18 and several DNA loci located on prophage regions, 17 i.e., BA5345, 21 BA5357, 46 and PL3. 47 Although most of these regions have been claimed to be anthrax-specific, B. cereus strains sometimes yield false positive results.…”

“…Assays mentioned by the World Health Organization (WHO) 31,40,44 were also included in the ring trial, as well as a hydrolysis probe assay 35 that targets the often used BA813 marker [31][32][33][34][35][36][37][38] (Table 3). The latter marker has shown in silico cross-reactions toward the near-neighbor strains in use in this trial and was included for this reason.…”

“…The most specific methods according to in silico analysis 21,46,47 were compared with the assays recommended by the WHO 40,44 and a single assay targeting BA813. 35 Ninety blinded DNA samples were exchanged between partners and an IAC was distributed. A detailed standard operative protocol describing how to conduct and perform the ring trial was set up after consultation of all participating laboratories.…”

Development of Three Real-Time PCR assays to Detect Bacillus anthracis and Assessment of Diagnostic Utility

“…Unless these PCRs have decreasing efficiencies (from pXO1 through BA chr) then this is most likely due to increased copy numbers per genome of the pXO1 plasmid over copy numbers of the pXO2 plasmid. A previous study [18] has indicated that copy number of the pXO1 plasmid can vary between 33-243 per bacterial cell, with pXO2 copy number varying between 1-32. The results of our study support these findings within the Ames DNA extract we used, also indicating that the pXO2 gene target has a higher copy number than that of the Ba chr PCR target.…”