16 research outputs found

    <i>FTL_0304</i>, <i>FTL_0291</i>, <i>FTL_0325</i> and <i>FTL_0057</i> mutants are highly attenuated for virulence in BALB/c and C57BL/6 mice.

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    a<p>6–8 weeks old mice were infected i.n. with either <i>F. tularensis</i> LVS or the indicated mutants and monitored for mortality for 28 days. Data are shown as number of mice survived/total number of mice infected.</p

    Immunization with the <i>FTL_0325</i> mutant induces an early pro-inflammatory cytokine response in lungs.

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    <p>BALB/c mice (n = 4) were infected with 1×10<sup>7</sup> CFU of the indicated mutants and wild type <i>F. tularensis</i> LVS. At the indicated times, the levels of pro-inflammatory cytokines were measured in lung homogenates using a Cytometric Bead Array assay. The data are representative of two independent experiments conducted and were analyzed using ANOVA with Tukey-Kramer Multiple Comparison post-test and <i>P</i> values were recorded. <i>**P</i><0.01; ***<i>P<</i>0.001. Ψ = Mice infected with 1×10<sup>7</sup> CFU of <i>F. tularensis</i> LVS succumbed to infection by day 7 PI and hence were unavailable for comparison.</p

    The <i>FTL_0325</i> and <i>FTL_0304</i> mutants of <i>F. tularensis</i> LVS exhibit no growth defect under acellular growth conditions.

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    <p>Growth curves for bacteria grown in MHB were generated in a 96-well plate using 200 µl culture volumes while the growth curves for bacteria grown in BHI and CDM were generated using a 25 ml culture volume. Results shown are representative of two independent experiments.</p

    Immunization with the <i>FTL_0325</i> mutant results in a potent memory recall response.

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    <p>Cell culture supernatants collected from BMDMs infected with either the <i>F. tularensis</i> LVS (A–D) or <i>F. tularensis</i> SchuS4 (E and F) and co-cultured with the splenocytes prepared from immunized mice at day 45 post-immunization (A and C) or at day 60 (B–F) were analyzed for IFN-γ (A, B and E) and IL-17a (C,D and F) cytokines by Cytometric Bead Flex sets. The data are representative of two independent experiments and were analyzed using ANOVA with Tukey-Kramer Multiple Comparison post-test and <i>P</i> values recorded. *<i>P<</i>0.05<i>; **P</i><0.01; ***<i>P<</i>0.001.</p

    Immunization with the <i>FTL_0325</i> mutant induces a higher pro-inflammatory cytokine response in the spleen.

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    <p>BALB/c mice (n = 4) were infected with 1×10<sup>7</sup> CFU of the indicated mutants and wild type <i>F. tularensis</i> LVS. At the indicated times, the levels of pro-inflammatory cytokines were measured in spleen homogenates using a Cytometric Bead Array assay. The data are representative of two independent experiments conducted and were analyzed using ANOVA with Tukey-Kramer Multiple Comparison post-test and <i>P</i> values were recorded. *<i>P<</i>0.05<i>; **P</i><0.01; ***<i>P<</i>0.001. Ψ = Mice infected with 1×10<sup>7</sup> CFU of <i>F. tularensis</i> LVS succumbed to infection by day 7 PI and hence were unavailable for comparison.</p

    Immunization with the <i>FTL_0325</i> mutant induces an early inflammatory response.

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    <p>BALB/c mice were infected with 1×10<sup>7</sup> CFU of the <i>F. tularensis</i> LVS, <i>FTL_0325</i> or <i>FTL_0304</i> mutant and the lungs were harvested at the indicated times, sectioned and stained with H & E. Representatives of H & E stained lung sections are shown. Infiltration of neutrophils is shown in the inset (Magnification 10×; Inset 100×). Ψ = Mice infected with 1×10<sup>7</sup> CFU of <i>F. tularensis</i> LVS succumbed to infection by day 7 PI and hence were unavailable for comparison. Green arrows indicate the sites of cellular infiltration. Yellow circles in inset show neutrophilic infiltration in the lungs of <i>FTL_0325</i> mutant immunized mice.</p

    Recall memory response results in an enhanced killing of <i>F. tularensis</i> SchuS4.

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    <p>The BMDMs were infected with <i>F. tularensis</i> SchuS4 and co-incubated with the splenocytes from naïve, <i>FTL_0325</i> or <i>FTL_0304</i> immunized mice. The BMDMs were lysed 72 hrs PI, diluted 10-fold and plated on MH-chocolate agar plates to determine the intracellular bacterial replication. The results were expressed as log<sub>10</sub> CFU/ml and are representative of two independent experiments. The data were analyzed using ANOVA with Tukey-Kramer Multiple Comparison post-test and <i>P</i> values were recorded. ***<i>P<</i>0.001.</p

    The <i>emrA1</i> mutant vaccinated mice induce sustained production of pro-inflammatory cytokines and a potent antibody response following lethal <i>Ft</i> LVS challenge.

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    <p>C57BL/6 mice immunized i.n. with 1×10<sup>6</sup> CFU of the <i>emrA1</i> mutant were challenged i.n. with 1×10<sup>7</sup> CFU of wild type <i>Ft</i> LVS 42 days post-immunization. <b>(A-D)</b> On days 5, 7 and 14 post-challenge, mice (n = 3 per group/time point) were euthanized and their excised lungs were homogenized. Clear lung homogenates were used for quantification of indicated pro-inflammatory cytokines using flow cytometric analysis. The data are represented as Mean ± S.D. <b>(E)</b> On day 21 post-challenge, mice (n = 3 per group) were anesthetized and bled retroorbitally to obtain serum. <i>Ft</i> specific total IgG, IgG2a, IgG2b, IgG1 and IgA levels in serum samples were determined by ELISA. The data are represented as Mean ± S.D. of absorbance values measured at 450 nm. Red arrows indicate antibody titers. ND = Not detected.</p

    Immunization with the <i>emrA1</i> mutant results in minimal weight loss, rapid bacterial clearance, and histopathological lesions in lung, liver and spleen.

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    <p>C57BL/6 mice were immunized i.n. with 1×10<sup>6</sup> CFU of the <i>emrA1</i> mutant. Mice infected with equal numbers of wild type <i>Ft</i> LVS were used as controls. <b>(A)</b> The immunized mice were weighed at the indicated times post-immunization to track the progress of infection. <b>(B)</b> On days 1, 5, 7, 14 and 21 post-immunization, mice (n = 4 per group/time point) were euthanized and bacterial burdens were quantified in their lung, liver and spleen. Bacterial counts in organs are expressed as Log<sub>10</sub>CFU/mL. The <i>P</i> values were determined using one way ANOVA. *<i>P<0</i>.<i>05; **P<0</i>.<i>01; ***P<0</i>.<i>001</i>. <b>(C)</b> Excised lungs, livers and spleens were preserved in 10% formalin, paraffin embedded, sliced into 5 μM thin sections and stained with Hematoxylene & Eosin. Stained sections were observed for histopathological lesions under a light microscope (Magnification 100×). # = <i>Ft</i> LVS infected mice succumbed to infection.</p
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