Improved Detection of Bartonella DNA in Mammalian Hosts and Arthropod Vectors by Real-Time PCR Using the NADH Dehydrogenase Gamma Subunit (nuoG) ▿

We used a whole-genome scanning technique to identify the NADH dehydrogenase gamma subunit (nuoG) primer set that is sensitive and specific enough to detect a diverse number of Bartonella species in a wide range of environmental samples yet maintains minimal cross-reactivity to mammalian host and arthropod vector organisms

Measurement of cytokines in supernatants of human PBMCs of immunized (group A) and naïve (group B) donors stimulated with recombinant Pla [5 μg/ml] or F1 [2 μg/ml].

<p>Group A was further divided into subgroups A-RV (recently vaccinated, n = 13) and A-EV (earlier vaccinated, n = 21) with post vaccination time less and more than one year, respectively. The SI was calculated against unstimulated cells (PBS). Concanavalin A (Con A) [1 mg/ml] served as positive control stimulus. The concentration of cytokines IL-10, and IL-17A (panel <b>A</b>) and IFN-γ, TNF-α, and IL-4 (panel <b>B</b>) is given in picograms per milliliter (pg/ml). The bars represent the median ± interquartile range calculated from quadruplicates. The statistical analysis was done by Mann-Whitney test. Statistically significant differences between the groups are indicated by * (<i>p</i><0.05), ** (<i>p</i><0.01), and *** <i>(p</i><0.0001).</p

Percent distribution of Pla-specific immunoglobulin classes and IgG subclasses among LPV vaccinated donors which were positive (A-Res) and negative (A-Non) in IgG ELISA with recombinant Pla as coating antigen.

<p>A-Total is combined group A, and naïve donors formed the group B. (A) IgG subclasses IgG1, IgG2, IgG3, and IgG4. (B) Antibody classes IgM, IgG, IgA, and IgE. Differences between the groups were statistically compared by the Chi-square test or the Fisher’s exact test when the number of samples was small. Statistically significant differences are indicated by * (<i>p</i><0.05) or by ** (<i>p</i><0.01).</p

Humoral and cellular immune responses to <i>Yersinia pestis</i> Pla antigen in humans immunized with live plague vaccine

<div><p>Background</p><p>To establish correlates of human immunity to the live plague vaccine (LPV), we analyzed parameters of cellular and antibody response to the plasminogen activator Pla of <i>Y</i>. <i>pestis</i>. This outer membrane protease is an essential virulence factor that is steadily expressed by <i>Y</i>. <i>pestis</i>.</p><p>Methodology/Principal findings</p><p>PBMCs and sera were obtained from a cohort of naïve (n = 17) and LPV-vaccinated (n = 34) donors. Anti-Pla antibodies of different classes and IgG subclasses were determined by ELISA and immunoblotting. The analysis of antibody response was complicated with a strong reactivity of Pla with normal human sera. The linear Pla B-cell epitopes were mapped using a library of 15-mer overlapping peptides. Twelve peptides that reacted specifically with sera of vaccinated donors were found together with a major cross-reacting peptide IPNISPDSFTVAAST located at the N-terminus. PBMCs were stimulated with recombinant Pla followed by proliferative analysis and cytokine profiling. The T-cell recall response was pronounced in vaccinees less than a year post-immunization, and became Th17-polarized over time after many rounds of vaccination.</p><p>Conclusions/Significance</p><p>The Pla protein can serve as a biomarker of successful vaccination with LPV. The diagnostic use of Pla will require elimination of cross-reactive parts of the antigen.</p></div