Characterization of an F1 Deletion Mutant of Yersinia pestis CO92, Pathogenic Role of F1 Antigen in Bubonic and Pneumonic Plague, and Evaluation of Sensitivity and Specificity of F1 Antigen Capture-Based Dipsticks

Abstract

We evaluated two commercial F1 antigen capture-based immunochromatographic dipsticks, Yersinia Pestis (F1) Smart II and Plague BioThreat Alert test strips, in detecting plague bacilli by using whole-blood samples from mice experimentally infected with Yersinia pestis CO92. To assess the specificities of these dipsticks, an in-frame F1-deficient mutant of CO92 (⌬caf) was generated by homologous recombination and used as a negative control. Based on genetic, antigenic/immunologic, and electron microscopic analyses, the ⌬caf mutant was devoid of a capsule. The growth rate of the ⌬caf mutant generally was similar to that of the wild-type (WT) bacterium at both 26 and 37°C, although the mutant's growth dropped slightly during the late phase at 37°C. The ⌬caf mutant was as virulent as WT CO92 in the pneumonic plague mouse model; however, it was attenuated in developing bubonic plague. Both dipsticks had similar sensitivities, requiring a minimum of 0.5 g/ml of purified F1 antigen or 1 ؋ 10 5 to 5 ؋ 10 5 CFU/ml of WT CO92 for positive results, while the blood samples were negative for up to 1 ؋ 10 8 CFU/ml of the ⌬caf mutant. Our studies demonstrated the diagnostic potential of two plague dipsticks in detecting capsular-positive strains of Y. pestis in bubonic and pneumonic plague.

Deletion of the Braun Lipoprotein-Encoding Gene and Altering the Function of Lipopolysaccharide Attenuate the Plague Bacterium

“…The capsular antigen (F1) of Y. pestis has antiphagocytic properties and is also a major immunoreactive antigen (39,46). Therefore, we examined the production of F1 antigen in all strains by using a commercial dipstick kit as well as by IF staining.…”

“…The release of RNase I was then measured as we previously described (44). For F1 production, the diluted cultures continued to grow at 37°C for an additional 2 h (as mentioned above) and then were analyzed with a commercially available plague detection kit, the Yersinia pestis (F1) Smart II kit (New Horizons Diagnostics, Baltimore, MD), as well as by immunofluorescence (IF) staining and microscopy as we previously described (46). Phagocytosis and intracellular survival assays.…”

Combinational Deletion of Three Membrane Protein-Encoding Genes Highly Attenuates Yersinia pestis while Retaining Immunogenicity in a Mouse Model of Pneumonic Plague

“…Samples from each flask were taken at 1-to 2-h intervals until the cultures reached their plateau phases. CFU were determined by plating (56). For visualization of membrane alterations, bacterial strains were grown to exponential phase at 28°C (optical density at 600 nm [OD 600 ] of 0.6).…”

“…For examining capsular antigen (F1) production by WT CO92 and its triple mutant, bacteria grown at 37°C were subjected to a commercially available plague detection kit, the Yersinia pestis (F1) Tetracore RedLine Alert kit (Tetracore, Rockville, MD), as we previously described (56). A ⌬caf1-negative mutant of CO92 devoid of F1 antigen was used as a control (56).…”

“…A ⌬caf1-negative mutant of CO92 devoid of F1 antigen was used as a control (56). We further analyzed F1 production by WT CO92 and its triple mutant by flow cytometry (56). Briefly, the above-mentioned Y. pestis cultures (10 6 CFU/sample) were fixed with 4% paraformaldehyde.…”