Histologic, serologic, and molecular analysis of persistent ehrlichiosis in a murine model

Abstract

Human monocytotropic ehrlichiosis caused by Ehrlichia chaffeensis was reported in 1987. An animal model to study acute fatal ehrlichiosis in mice that has been developed closely resembles the fatal form of human monocytotropic ehrlichiosis. However, animal models for persistent infection in the genus Ehrlichia in immunocompetent mice have not been characterized. We report the histopathological progression of Ehrlichia muris infection in immunocompetent mice (AKR and C57BL/6 strains) correlated with their antibody response determined by indirect immunofluorescence and Western immunoblotting, and the distribution and quantity of the ehrlichial load by immunohistochemistry, polymerase chain reaction (PCR), and real-time PCR in lungs, liver, and spleen. Mild to moderate correlation was observed between histopathological grading in these organs and relative ehrlichial loads. The highest ehrlichial loads were present between days 4 and 14 after infection. E. muris was detected in tissues examined up to 150 days after infection by real-time PCR. Analysis of the serological response revealed several immunodominant antigens, including 200-, 180-, 100-, 73/75-, 45-, and 28-kd proteins. In conclusion, we have provided for the first time a complete histopathological, serological, immunohistochemical, and quantitative analysis of an animal model for the study of persistent ehrlichial infection.

Figure 1

A: Liver at 9 days after infection. LHI in the hepatic lobule and Kupffer cell hyperplasia (H&E). B: Liver at 30 days after infection. Prominent perivascular infiltrates with well-formed granulomas (arrows). Kupffer cell hyperplasia (H&E). C: Liver, day 14 after infection. Ehrlichial morulae in sinusoidal mononuclear cells (horseradish peroxidase/diaminobenzidine). D: Lung, day 9 after infection. Focal LHI (arrow) in the lung interstitium (H&E). E: Lung, day 20 after infection. Interstitial, perivascular (arrow), and peribronchial (arrow) LHI (H&E). F: Lung, day 14 after infection. Ehrlichial morulae in mononuclear cells infiltrating the lung interstitium (horseradish peroxidase/diaminobenzidine). G: Section of spleen at day 4 after infection. Infiltration of red pulp and marginal zone by macrophages (H&E). H: Spleen section at day 14 after infection. Markedly irregular lymphoid follicles and macrophage infiltration of red pulp (H&E). I: Spleen at day 10 after infection. Ehrlichial morulae in macrophages infiltrating red pulp (horseradish peroxidase/diaminobenzidine). Original magnifications: ×200 (A, B, D); ×1000 (C, F, I); ×100 (E, G, H).

Figure 2

Western immunoblots demonstrating the kinetics of antibody responses (IgG) to E. muris whole-cell lysates in pooled sera from two strains of mice (C57BL/6, A; AKR, B) experimentally infected intraperitoneally with live E. muris. Serum samples were collected at time points up to 120 days after inoculation as indicated above each corresponding lane. Molecular mass is shown in kilodaltons (15 to 250 kd).