Pathologic evaluation of Syzygium aromaticum essential oil against pulmonary aspergillosis in non-neutropenic immunosuppressed rabbits

Aspergillus is a genus of saprophytic fungi which is found in soil, construction equipment, and hospital environments. Invasive aspergillosis occurring in immunocompromised individuals or in patients receiving immunosuppressive therapy is recognized as a major cause of mortality. In the in vitro phase of this study, clove essential oil was prepared by hydrodistillation of clove plant (Syzygium aromaticum) dry powder, GC-MS analysis followed by the MIC and MFC determination of clove essential oil were performed. In the in vivo phase, 24 female New Zealand White rabbits were exposed to a conidial suspension of Aspergillus fumigatus ATCC 13,073 via the endotracheal route after administration of non-neutropenic immunosuppressive agents (cyclosporine and methylprednisolone). The rabbits were then assigned into four groups: normal saline, Itraconazole, clove essential oil at 20 mg/kg, and clove essential oil at 10 mg/kg, administered orally every 24 h. Daily monitoring of the rabbits was performed, and CT scans were conducted on days 0, 7, and 13 post-inoculations. On days 7 and 13 post-inoculation, half of the animals were sacrificed. Following this, Macroscopic examinations and sampling were conducted to evaluate bronchoalveolar lavage fluid and lung tissue fungal burden. For microscopical examinations of pulmonary lesions was used H&E staining, immunohistochemistry markers (CD68, CD163, and MPO), and Grocott's methenamine silver (GMS) staining. Systemic toxicity in other vital organs was also assessed using H&E staining. The clove essential oil, containing 96.81% eugenol, demonstrated acceptable MIC and MFC activity for Aspergillus fumigatus ATCC 13,073, comparable to Itraconazole. In both pre-mortem and post-mortem evaluations, the Itraconazole group and the clove essential oil group at a dose of 20 mg/kg exhibited significant reductions in fungal burden, fungal infection, and pulmonary involvement, without notable systemic toxicity changes in other tissues. Clove essential oil exhibited acceptable efficacy comparable to a commercial azole antifungal agent under both in vitro and in vivo conditions, effectively reducing the fungal burden of Aspergillus fumigatus ATCC 13,073 and associated pulmonary lesions without inducing systemic toxicity.