Murine models of scrub typhus associated with host control of <i>Orientia tsutsugamushi</i> infection

<div><p>Background</p><p>Scrub typhus, a febrile illness of substantial incidence and mortality, is caused by infection with the obligately intracellular bacterium <i>Orientia tsutsugamushi</i>. It is estimated that there are more than one million cases annually transmitted by the parasitic larval stage of trombiculid mites in the Asia-Pacific region. The antigenic and genetic diversity of the multiple strains of <i>O</i>. <i>tsutsugamushi</i> hinders the advancement of laboratory diagnosis, development of long-lasting vaccine-induced protection, and interpretation of clinical infection. Despite the life-threatening severity of the illness in hundreds of thousands of cases annually, 85–93% of patients survive, often without anti-rickettsial treatment. To more completely understand the disease caused by <i>Orientia</i> infection, animal models which closely correlate with the clinical manifestations, target cells, organ involvement, and histopathologic lesions of human cases of scrub typhus should be employed. Previously, our laboratory has extensively characterized two relevant C57BL/6 mouse models using <i>O</i>. <i>tsutsugamushi</i> Karp strain: a route-specific intradermal model of infection and persistence and a hematogenously disseminated dose-dependent lethal model.</p><p>Principal findings</p><p>To complement the lethal model, here we illustrate a sublethal model in the same mouse strain using the <i>O</i>. <i>tsutsugamushi</i> Gilliam strain, which resulted in dose-dependent severity of illness, weight loss, and systemic dissemination to endothelial cells of the microcirculation and mononuclear phagocytic cells. Histopathologic lesions included expansion of the pulmonary interstitium by inflammatory cell infiltrates and multifocal hepatic lesions with mononuclear cellular infiltrates, renal interstitial lymphohistiocytic inflammation, mild meningoencephalitis, and characteristic typhus nodules.</p><p>Significance</p><p>These models parallel characteristics of human cases of scrub typhus, and will be used in concert to understand differences in severity which lead to lethality or host control of the infection and to address the explanation for short duration of heterologous immunity in <i>Orientia</i> infection.</p></div

Body and spleen weight change of mice inoculated intravenously or intradermally with <i>O</i>. <i>tsutsugamushi</i> Gilliam strain.

<p>Percent body weight change (<b>A</b>) or spleen weight in milligrams (<b>B</b>) of animals inoculated intravenously with either 7.5x10⁶ (high dose, <b>circles</b>), 7.5x10⁵ (mid-dose, <b>squares</b>), 7.5x10³ (low dose, <b>triangles</b>), or intradermally with 2.5x10⁵ organisms (<b>asterisks</b>) as compared to sham inoculated control (<b>diamonds</b>). **, <i>p</i><0.01, ***, <i>p</i><0.001.</p

Inhibition of filopodium formation prevented <i>Ehrlichia</i> intercellular transport.

<p>(A) <i>Ehrlichia-</i>infected DH82 cells were treated with CFSE and seeded with uninfected non-treated DH82 cells for 24 hours. Thick arrow indicates <i>E. muris</i> (probed with <i>Ehrlichia</i> HSP60), whereas the thin arrows indicate filopodia/pseudopodia of infected cells. DAPI stains the nucleus of both the uninfected and infected cells. The adjacent figure is the Nomarski image, which clearly showed the filopodia/pseudopodia of infected cells. (B, C) DH82 cells were treated with CFSE and seeded with infected non-treated DH82 cells for 24 hours. Thick arrow indicates <i>E. muris</i> (probed with <i>Ehrlichia</i> HSP60) whereas the thin arrows indicate filopodia/pseudopodia of infected cells. DAPI stains the nuclei of both uninfected and infected cells. The adjacent figure is the Nomarski image which showed clearly the filopodia/pseudopodia of infected cells. (D) <i>Ehrlichia-</i>infected DH82 cells were treated with CFSE and seeded with uninfected non-treated DH82 cells for 24 hours in the presence of cytochalasin D. Thick arrow indicates <i>E. muris</i> (the adjacent figure is the Nomarski image). (E) Uninfected DH82 cells were treated with CFSE and seeded with infected non-treated DH82 cells for 24 hours in the presence of cytochalasin D. Thick arrow indicates <i>E. muris</i> (the adjacent figure is the Nomarski image). (F). Quantitative real time-PCR of bacterial loads of <i>E. muris-</i>infected DH82 cells to evaluate cytotoxicity in the presence of cytochalasin D (n = 3 per group). (G). Quantitative real time-PCR of bacterial load of <i>E. muris-</i> infected DH82 cells seeded with uninfected DH82 cells in the presence and absence of cytochalasin D (n = 3 per group).</p

Location <i>of O</i>. <i>tsutsugamushi</i> Gilliam antigens following i.v. or i.d. inoculation.

<p>Sections of the lungs at 9 dpi after i.v. inoculation reveal the presence of <i>Orientia</i> antigens (red) in interstitial capillary vessels and alveolar septa (<b>A</b>). Orientia antigens co-localize with splenic (<b>B</b>) and hepatic (<b>C</b>) macrophages. Orientia antigen in a cerebral vessel surrounded by a characteristic typhus nodule 18 dpi after i.d. inoculation (<b>D</b>, 400X, inset 1,000X, bars = 100 μm).</p

Dose-dependent severity of histopathologic renal lesions and lung pathology in mice inoculated i.v. or i.d. with <i>O</i>. <i>tsutsugamushi</i> Gilliam strain.

<p>Representative histopathologic renal inflammatory infiltrates between tubules of the renal cortex (arrows) at 6 dpi after i.d. inoculation (<b>A</b>, left) and i.v. mid-dose at 15 dpi (<b>A</b>, right, 100X). Renal inflammatory index (<b>B</b>) or lung pathology score (<b>C</b>) after i.v. inoculation with high (<b>circles</b>), mid (<b>squares</b>) low (<b>triangles</b>) dose or i.d. (<b>asterisks</b>) with <i>O</i>. <i>tsutsugamushi</i>. *, <i>p</i><0.05, **, <i>p</i><0.01, ***, <i>p</i><0.001. Asterisks with bars indicate data points statistically different from baseline.</p

<i>Ehrlichia</i> are contained in the filopodia of DH82 cells.

<p>(A) Filopodia extended from the polar ends of the <i>E. chaffeensis-</i> infected DH82 cell. Left: <i>E. chaffeensis-</i>infected DH82 cell probed with anti-Hsp60 antibody. Thick arrow indicates <i>E. chaffeensis</i> intracellular colonies and thin arrow indicates filopodium. Middle: <i>E. chaffeensis-</i>infected cell stained with DAPI. Thick arrow indicates morulae of <i>E. chaffeensis</i> stained with DAPI and thin arrow indicates host nucleus. Right: Merged figure. Scale bar, 25 micrometers. (B) Filopodia of <i>E. chaffeensis-</i> infected DH82 cells extended to neighboring cells. (C) When host cells were not in the immediate vicinity, the leading edge of an <i>E. chaffeensis-</i> infected DH82 cell formed a flattened fan-shaped structure filled with the pathogen. (D) Uninfected DH82 cell. (E) Uninfected DH82 cell stained with Diff-Quik stain. (F) <i>E. muris-</i>infected DH82 cell stained with Diff-Quik stain. Scale bar, 25 micrometers. (G) Transmission electron micrograph of a DH82 cell infected with <i>E. muris</i>. Arrows indicate morulae of <i>E. muris</i>. Scale bar, 1 micrometer.</p

Development of antibodies to <i>O</i>. <i>tsutsugamushi</i> Gilliam strain following challenge.

<p>Development of antibodies to <i>O</i>. <i>tsutsugamushi</i> Gilliam strain following challenge.</p

Bacterial dissemination after infection with <i>O</i>. <i>tsutsugamushi</i> Gilliam strain.

<p>Bacterial loads in spleen (<b>A</b>), lung (<b>B</b>), liver (<b>C</b>) and kidney (<b>D</b>) after i.v. inoculation with high (<b>circles</b>), mid (<b>squares)</b>, or low dose (<b>triangles</b>), or via i.d. inoculation <b>(asterisks)</b>.</p

<i>Ehrlichia</i> morulae inside a DH82 cell with an overlying ruptured host cell membrane.

<p>(A) Different stages of <i>E. muris</i> in a mechanically opened DH82 cell. Thin arrow indicates dividing ehrlichiae; thick arrow indicates mature cells. (B) Mature ehrlichiae cells in a large morula (thick arrow). (C) Pore formation on a DH82 host cell containing many ehrlichiae that have deformed the overlying cell membrane (thin arrows) (intact DH82 cell). (D) Host cell membrane ruptured at the location of ehrlichial exit from the cell (intact DH82 cell). (E) Extracellular <i>Ehrlichia</i> attached with high affinity to the filopodium of neighboring host cells (thin arrows). (F) Ehrlichiae attached to the DH82 cell membrane adjacent to a cell membrane ruffle (thin arrow) (intact DH82 cell). (G) TEM of an IOE infected spleen (arrows indicate fused morula).</p

<i>Ehrlichia</i> are observed in the filopodia of mouse macrophages.

<p>(A) <i>E. muris-</i>infected mouse macrophages probed with <i>Ehrlichia</i> Hsp60 antibody (left), DAPI (middle) (thick arrows indicate DNA of <i>E. muris,</i> and thin arrows indicate mouse macrophage nuclei), and merged figure (right). (B) IOE-infected mouse macrophage probed with <i>Ehrlichia</i> Hsp60 antibody (left) (thin arrow indicates filopodium), DAPI (middle), and merged figure (right). (C) Scanning electron micrograph of <i>E. muris-</i>induced filopodium in a mouse macrophage; thin arrow indicates the filopodium. (D) The interior of a mouse macrophage from which the cell membrane has been removed contained <i>E. muris</i>. (E) Higher magnification of <i>E. muris</i> in a mouse macrophage. (F) Scanning electron micrograph of an <i>E. muris</i> bacterium. (G, H) Scanning electron micrograph of IOE-induced filopodia in mouse macrophages; thin arrows indicate the filopodia. (I) IOE microorganisms in a mouse macrophage. (J) Scanning electron micrograph of a single IOE bacterium. (K) Uninfected mouse macrophage. (L) High magnification of an opened uninfected mouse macrophage. (M) Transmission electron micrograph of a mouse macrophage that contained an <i>E. muris</i> morula (thick arrow), N, nucleus. Scale bar, 1 micrometer.</p