Interstitial pneumonia via the oropharyngeal route of infection with Encephalitozoon cuniculi Oropharyngeal infection with Encephalitozoon cuniculi

Microsporidia causes opportunistic infections in immunosuppressed individuals. Mammals shed these spores of fungi in feces, urine, or respiratory secretions, which could contaminate water and food, thereby reaching the human body and causing infection. The oral route is the most common route of infection, although experiments have demonstrated that intraperitoneal and intravenous routes may also spread infection. Respiratory tract infection, although considered to be possible, has not been reported to date. The present study, therefore, aimed to demonstrate infection with the microsporidia of Encephalitozoon cuniculi via the oropharyngeal route as a model for opportunistic pneumonia. The objectives were the study of opportunistic pneumonia in general while also confirming transmission via the respiratory route to expand the understanding of the epidemiology of zoonoses. C57BL mice, both male and female, up to 12 weeks of age, and free of specific pathogens (SPF) were inoculated (Infected group) or not infected (Non-Infected group) with 1 × 10 ⁷ spores of E. cuniculi via the oropharyngeal route. The animals immunosuppressed with cyclophosphamide (Cy) were infected (Cy-Infected group) or not infected (Cy-Uninfected group), and then assessed for the influence of immunosuppression on infection. In both groups, animals inoculated with 0.9% saline solution via the oropharyngeal route served as controls (Sham and Cy-Sham groups, respectively). After 14 days of infection, the lungs of all animals were retrieved for histopathological analysis, phenotyping of lung inflammatory cells using flow cytometry, measurement of fungal load using qPCR, and measurement of the serum levels of Th1, Th2, and Th17 cytokines. The results revealed that the infected animals developed interstitial pneumonia characterized by perialveolar inflammatory infiltrative lesions with a predominance of lymphocytes and plasma cells. However, the fungal load and the extent of the inflammatory infiltrate were relatively lower in the Cy-Infected group, with a predominance of the CD8 ⁺ T lymphocyte population in the lungs compared to Infected group. In the Infected group not treated with Cy, an increase in the population of alveolar macrophages (F4/80 ⁺ CD11b ^- SiglecF ⁺ ) was noted, along with higher fungal load and inflammatory infiltrate in the lungs, indicating a further pronounced Encephalitozoon pneumonia compared to the immunosuppressed animals. The infected groups presented Th1 cytokine profiles, with the Cy-Infected group exhibiting increased Th17 levels. Collectively, these results demonstrated that oropharyngeal infection promoted pneumonia caused by E. cuniculi in mice treated or not with cyclophosphamide, with greater severity occurring in the non-immunosuppressed mice, thereby establishing this model as a suitable one for interstitial pneumonia via aspiration.