Murine models of scrub typhus associated with host control of Orientia tsutsugamushi infection

Abstract

Background: Scrub typhus, a febrile illness of substantial incidence and mortality, is caused by infection with the obligately intracellular bacterium Orientia tsutsugamushi. 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 O. tsutsugamushi 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 Orientia 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 O. tsutsugamushi Karp strain: a route-specific intradermal model of infection and persistence and a hematogenously disseminated dose-dependent lethal model.

Principal findings: To complement the lethal model, here we illustrate a sublethal model in the same mouse strain using the O. tsutsugamushi 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.

Significance: 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 Orientia infection.

Fig 1. Body and spleen weight change of mice inoculated intravenously or intradermally with O. tsutsugamushi Gilliam strain.

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

Fig 2. Hematologic responses to O. tsutsugamushi challenge.

Circulating absolute lymphocyte (A) and neutrophil concentrations (B), percent hematocrit (C) and platelet counts (D) of uninfected (n = 5, diamonds) mice or those inoculated (n = 5) intravenously with high dose (circles) mid-dose (squares) low dose (triangles) or via intradermal route (asterisks) Grey shading represents murine normal range. *, p<0.05.

Fig 3. Seroconversion after infection with O. tsutsugamushi Gilliam strain.

Reciprocal endpoint IgG titer of mice as determined by indirect IFA after i.v. inoculation with high (A), mid (B), or low dose (C) or via the intradermal route (D). The IgG isotype response was further categorized into IgG1 (E) or IgG2c (F) at the final time point, 15 dpi for the i.v. route and 30 dpi for the i.d. route. Serum which was nonreactive at a 1:64 titer is represented by a value of zero.

Fig 4. Bacterial dissemination after infection with O. tsutsugamushi Gilliam strain.

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

Fig 5. Dose-dependent liver inflammatory index of mice inoculated i.v. or i.d. with O. tsutsugamushi Gilliam strain.

Liver inflammatory index after i.v. inoculation with high (A), mid (B) or low (C) dose or i.d. (D) with O. tsutsugamushi. ***, p<0.001.

Fig 6. Dose-dependent severity of histopathologic renal lesions and lung pathology in mice inoculated i.v. or i.d. with O. tsutsugamushi Gilliam strain.

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

Fig 7. Location of O. tsutsugamushi Gilliam antigens following i.v. or i.d. inoculation.

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