<i>R</i>. <i>conorii</i> (ISF) induced significant secretion of IL-8 and IL-6, while <i>R</i>. <i>massiliae</i> induced significant production of MCP-1 in infected HMEC-1 cells.

<p>The production levels of MCP-1 (A), IL-8 (B) and IL-6 (C) in the supernatant of <i>Rickettsia</i>-infected HMEC-1 cells was assessed by ELISA. Bar graphs indicate the average and standard error of three independent experiments. White bars represent uninfected cells, grey bars represent <i>R</i>. <i>massiliae</i>-infected cells, and <i>R</i>. <i>conorii</i> (ISF)—infected cells are represented by black bars. *, <i>p</i> < 0.05, ** n.s. = non-statistically significant.</p

<i>R</i>. <i>massiliae</i> and <i>R</i>. <i>conorii</i> (ISF) infection resulted in plaques in Vero cell monolayers and replicated efficiently in the human dermal microvascular cell line, HMEC-1 cells.

<p>(A) <i>R</i>. <i>massiliae</i> and <i>R</i>. <i>conorii</i> (ISF) were grown in Vero cells at 34°C for 5 and 7 days to quantify the number of plaques present as revealed by crystal violet staining (B). <i>R</i>. <i>massiliae</i> and <i>R</i>. <i>conorii</i> (ISF) were grown in HMEC-1 cells for times indicated. Data are representative of three independent experiments (A and B).</p

<i>R</i>. <i>massiliae</i> induced upregulation of MCP-1 mRNA levels and <i>R</i>. <i>conorii</i> (ISF) induced IL-8 mRNA levels in HMEC-1 cells.

<p>Real-time quantitative PCR analysis of the expression of MCP-1 mRNA (A) and IL-8mRNA (B) in HMEC-1 cells infected at an MOI of 5. Bar graphs indicate the average and standard error of three independent experiments. White bars represent uninfected cells, grey bars represent <i>R</i>. <i>massiliae-</i> infected cells, and <i>R</i>. <i>conorii</i> (ISF)-infected cells are represented by black bars. *, <i>p</i> <0.05.</p

<i>R</i>. <i>conorii</i> (ISF), but not <i>R</i>. <i>massiliae</i>, increased endothelial cell monolayer permeability.

<p>(A) Representative ECIS graph demonstrating the loss of electrical resistance across an HMEC-1 monolayer in real-time. (B) Average resistance of endothelial cell monolayers after infection with rickettsiae for designated time points. Bar graphs (B) indicate the average and standard error of three independent experiments. White bars represent uninfected cells, grey bars represent <i>R</i>. <i>massiliae</i>-infected cells, and <i>R</i>. <i>conorii</i> (ISF)—infected cells are represented by black bars. *, <i>p</i> < 0.05.</p

<i>R</i>. <i>conorii</i> (ISF), but not <i>R</i>. <i>massiliae</i>, induced cell death at 72 HPI in HMEC-1 cells: (A) Cells were infected at an MOI of 5 and stained with Live/Dead fixable dye for 30 minutes before fixation in paraformaldehyde.

<p>For flow cytometry experiments, ⩾10,000 cells were analyzed. (B) Annexin V staining of HMEC-1 cells following 72 hours of infection with <i>R</i>. <i>massiliae</i> or <i>R</i>. <i>conorii</i> (ISF). Cell counts were normalized to mode. (C) Lactate dehydrogenase (LDH) activity assay of monolayer supernatants demonstrating significantly increased levels of endothelial cell cytotoxicity after infection with <i>R</i>. <i>conorii</i> (ISF) for 72 hours. Bar graphs (A and C) indicate the average and standard error of three independent experiments. White bars represent uninfected cells, grey bars represent <i>R</i>. <i>massiliae</i>-infected cells, <i>R</i>. <i>conorii</i> (ISF)-infected cells are represented by black bars, and cells treated with staurosporine are represented by the checkered bar. *, <i>p</i> < 0.05.</p

Endothelial cell death induced by <i>R</i>. <i>conorii</i> (ISF) is partially dependent on caspase-1.

<p>Cells were treated with caspase-1 and caspase-4 inhibitors and then infected with <i>R</i>. <i>conorii</i> (ISF) for 72 hours. Fresh medium and caspase inhibitors were added daily, and the removed supernatant was used immediately for the LDH activity assay. The bar graph indicates the average and standard error of three independent experiments. *, <i>p</i> < 0.05, inh = inhibitor, Rc = <i>R</i>. <i>conorii</i> (ISF), casp = caspase.</p

DataSheet_1_Case Report: Metagenomic next-generation sequencing applied in diagnosing psittacosis caused by Chlamydia psittaci infection.docx

BackgroundChlamydia psittaci is the causative agent of psittacosis in humans, while its rapid identification is hampered due to the lack of specificity of laboratory testing methods.Case presentationThis study reports four cases of C. psittaci infection after contact with a domestic parrot, all belonging to the same family. Common manifestations like fever, cough, headache, nausea, and hypodynamia appeared in the patients. Metagenomic next-generation sequencing (mNGS) aided the etiological diagnosis of psittacosis, revealing 58318 and 7 sequence reads corresponding to C. psittaci in two cases. The detected C. psittaci was typed as ST100001 in the Multilocus-sequence typing (MLST) system, a novel strain initially reported. Based on the results of pathogenic identification by mNGS, the four patients were individually, treated with different antibiotics, and discharged with favorable outcomes.ConclusionIn diagnosing psittacosis caused by a rare C. psittaci agent, mNGS provides rapid etiological identification, contributing to targeted antibiotic therapy and favorable outcomes. This study also reminds clinicians to raise awareness of psittacosis when encountering family members with a fever of unknown origin.</p

Image_1_Case Report: Metagenomic next-generation sequencing applied in diagnosing psittacosis caused by Chlamydia psittaci infection.tif

BackgroundChlamydia psittaci is the causative agent of psittacosis in humans, while its rapid identification is hampered due to the lack of specificity of laboratory testing methods.Case presentationThis study reports four cases of C. psittaci infection after contact with a domestic parrot, all belonging to the same family. Common manifestations like fever, cough, headache, nausea, and hypodynamia appeared in the patients. Metagenomic next-generation sequencing (mNGS) aided the etiological diagnosis of psittacosis, revealing 58318 and 7 sequence reads corresponding to C. psittaci in two cases. The detected C. psittaci was typed as ST100001 in the Multilocus-sequence typing (MLST) system, a novel strain initially reported. Based on the results of pathogenic identification by mNGS, the four patients were individually, treated with different antibiotics, and discharged with favorable outcomes.ConclusionIn diagnosing psittacosis caused by a rare C. psittaci agent, mNGS provides rapid etiological identification, contributing to targeted antibiotic therapy and favorable outcomes. This study also reminds clinicians to raise awareness of psittacosis when encountering family members with a fever of unknown origin.</p

CpG-B-induced death of <i>R. australis</i>-infected mice is independent of pDC.

<p>Depletion of pDC was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034062#s2" target="_blank"><i>Methods</i></a>. At day 2 after infection with <i>R. australis</i> (5×10⁵ pfu), 50 µg of CpG-B per mouse were injected i.v. into control or pDC-depleted mice. Mouse survival (6–8 mice per group) was monitored for 14 days.</p

Multiple intracellular cytokines produced by CD4⁺ and CD8⁺ T cells.

<p>At day 2 after infection with 5×10⁵ pfu of <i>R. australis</i>, 50 µg of ODN control (ODN 1826 control) and CpG-B (ODN 1826) per mouse were injected i.v. into WT and IDO^−/− mice (4 mice per group). At day 5 post-infection, spleen cells (1×10⁶/ml) from individual mice were restimulated with PMA/Ionomycin/GolgiStop for 5 h. Multiple intracellular cytokines as indicated were measured in CD4⁺ and CD8⁺ T cells. The percentages of intracellular cytokine production in gated T cells were shown as mean ± SD in the corner (A). Representative statistical data are shown from one of three independent experiments. * <i>p</i><0.05 (B).</p