Introduction

Aspergillus, a ubiquitous saprophytic fungus, is widely found in soil, building dust, and hospital environments1. Aspergillus fumigatus spores continually threaten the respiratory tract by inducing inflammatory responses predominantly mediated by macrophages and epithelial cells2. Pulmonary aspergillosis is generally classified into three broad categories: allergic bronchopulmonary aspergillosis, chronic pulmonary aspergillosis, and invasive pulmonary aspergillosis1. Invasive aspergillosis (IA) is recognized as a significant cause of morbidity and mortality in severely immunocompromised patients and primarily involves the lungs through inhalation of Aspergillus conidia3. Consequently, invasive pulmonary aspergillosis (IPA) is considered a serious and life-threatening infection in patients undergoing immunosuppressive therapies with severe and prolonged neutropenia, such as myelotoxic chemotherapy for cancer treatment4. Invasive pulmonary aspergillosis progresses rapidly and has a high mortality rate of up to 50%1. The critical role of neutrophils in eliminating Aspergillus conidia has been proven1,5. Additionally, the efficacy of lysosomes in phagosomes and the acidification of phagosomes determine the capacity of airway epithelial cells to control Aspergillus fumigatus growth. Overall, the airway epithelium demonstrates greater ability to inhibit fungal survival in bronchial cells in comparison with alveolar cells, and specific cell wall mutants of Aspergillus fumigatus can affect phagosome maturation in epithelial cells6. Recent years have witnessed an increased incidence of invasive aspergillosis in non-neutropenic patients, including individuals with hematological malignancies undergoing chemotherapy, patients admitted to intensive care unit (ICU), and those treated with corticosteroids were reported1,7. It is estimated that only one-third of IPA cases are associated with long-term neutropenia1. Moreover, the emergence of COVID-19-associated pulmonary aspergillosis in ICU patients has further highlighted the importance of awareness regarding this pathogen8. Due to the clinical significance of this disease, extensive research is conducted annually on its pathophysiology, epidemiology, prognostic factors, and treatment methods9,10.

Immunosuppressive therapies, such as cyclosporine administration, are major predisposing factors for IPA4. Cyclosporine, a potent immunosuppressant, is commonly used to prevent organ transplant rejection and manage autoimmune disorders11. It inhibits T-cell activation, which, while effective in preventing graft-versus-host disease, significantly impairs the immune system’s capacity to combat infections. As a result, patients receiving cyclosporine are at heightened risk for opportunistic infections, with Aspergillus representing one of the most prevalent and life-threatening pathogens11. Histopathological evaluation is a crucial tool for studying IPA, particularly for assessing lung tissue damage and disease progression in immunocompromised hosts. Examination of lung tissues under a light microscope allows the identification of pathological changes, including tissue necrosis, inflammation, and fungal hyphae presence all of which are indicative of Aspergillus invasion. Such analysis provides insights into fungal infection mechanisms and host responses, which are essential for developing effective diagnostic and therapeutic strategies9,10. Within ICU, the revised EORTC/MSG definitions categorize IPA in immunocompromised patients as proven, probable, or possible. Proven IPA requires histopathological confirmation of fungal invasion; probable IPA necessitates a combination of host factors, clinical signs, and positive mycological evidence, and possible IPA is identified by host factors and clinical signs but lacks mycological confirmation12,13.

Many clinically-used antifungal agents are associated with toxicity, drug–drug interactions, lack of fungicidal effect, high cost, and emerging resistance among common fungal pathogens14. Therefore, extensive studies have been performed on Aspergillus both in vitro and in vivo15. Recent treatment recommendations have been updated emphasizing the combination of first line and second-line therapies, alongside the investigations into novel compounds16. Since Aspergillus species are well-known to be resistant to azole antifungal compounds, plant-derived essential oils and their major constituents have been evaluated for antifungal activity17. Clove plant (Syzygium aromaticum), a member of the Myrtaceae family, is globally among the most valued spices18,19. This plant contains eugenol, acetyl eugenol, beta-caryophyllene, vanillin, keratolic acid, tannins, and other compounds19. Eugenol, - a volatile, colorless to light-yellow, water-soluble phenylpropanoid with a strong odor and taste- constitutes the primary component of clove essential oil. It exhibits insecticidal, antimicrobial, and anti-inflammatory properties20 and demonstrates potent antifungal activity against A. fumigatus in in vitro studies14,17,21,22. In addition to in vitro studies, the establishment of animal models of pulmonary aspergillosis and the design of preclinical studies to investigate antifungal are of great importance15,23,24. Rabbits in particular serve as valuable models due to their susceptibility to respiratory infections and similarities in immune response to humans15,24,25. Pulmonary lesions in immunosuppressed rabbits are classified into three categories: hemorrhagic infarction, neutrophilic, and monocytic lesions24,25. The use of cyclosporine–methylprednisolone-treated rabbits simulate a non-neutropenic immunocompromised state, providing a relevant model for IPA studies15.

This study aimed to establish an animal model of invasive pulmonary aspergillosis using standard non-neutropenic immunosuppression methods15,24,26, and to evaluate the antifungal efficacy of clove essential oil in comparison with a conventional antifungal agent regarding in vitro and in vivo aspects. Therefore, the effects as well as therapeutic potency of clove essential oil at two different doses compared to Itraconazole in the direct exposure to fungus Aspergillus fumigatus ATCC 13,073 along with treatment of pulmonary aspergillosis in rabbits were investigated clinically, paraclinically, necropsy, mycologically and histopathologically.

Materials and methods

Fungal strain and inoculum preparation

A standard isolate of Aspergillus fumigatus (ATCC 13073) was used in this study. The cryopreserved stock culture was first thawed at room temperature. To activate the fungus, it was sub-cultured onto potato dextrose agar (PDA) plates and incubated at 28 °C for 7 days to promote extensive sporulation and conidia formation27,28. The conidia were then harvested by gently flooding the agar surface with sterile saline containing 0.05% Tween 80. The resulting suspension was filtered through sterile gauze to remove hyphal fragments, and the conidial concentration was adjusted using a hemocytometer slide28.

Extraction and chemical analysis of essential oil

Clove essential oil was extracted from 100 g of dried Syzygium aromaticum (clove) powder via hydrodistillation for 4 h using a Clevenger-type apparatus. The collected oil was dehydrated over anhydrous sodium sulfate and stored in an amber-glass vial at 4 °C to protect it from light and oxidation until further use19.

The chemical composition of the essential oil was characterized by gas chromatography–mass spectrometry (GC–MS). The analysis was performed using Thermoquest 2000. The chromatograph was equipped with Hp5 capillary column (30 × 0.25 mmID× 0.25 μm film thickness) and the data were acquired under the following conditions: initial temperature 50 °C, program rate 2.5 °C, final temperature 265 °C and injector temperature 250 °C. The carrier gas was helium at the flow rate of 1.1 mL/min, and the split ratio was 1:100. The essential oil was also analysed by GC mass spectrometry (GC/MS) on the same gas chromatograph coupled with MSD5973. The MS was run in the electron ionization mode, using ionization energy of 70 eV and interface temperature was 250 °C. The components of essential oils were identified tentatively by comparing their retention indices and mass spectra with those of Wiley 275 Registry of Mass Spectral Data. The relative percentages of the constituents were calculated based on peak areas19.

Determination of minimum inhibitory and fungicidal concentrations (MIC/MFC)

The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of clove essential oil against Aspergillus fumigatus (ATCC 13073) were determined according to the reference broth microdilution method27. The fungal strain was sub-cultured from a frozen stock onto Sabouraud dextrose agar (SDA) and incubated at 28 °C for 5–7 days to promote conidiation. Conidia were harvested by adding 5 mL of sterile saline with 0.05% Tween 80 to the agar surface and gently scraping with a sterile loop. The resulting suspension was vortexed, and the conidial density was adjusted to a concentration of 4 × 10⁴ CFU/mL in Roswell Park Memorial Institute medium (RPMI) 1640 medium buffered with MOPS, as confirmed by hemocytometer count. Two-fold serial dilutions of the clove essential oil were prepared in RPMI 1640 medium, yielding a concentration range of 0.02 to 20 µL/mL. Aliquots of 100 µL of each dilution were dispensed into the wells of a sterile 96-well microtiter plate. An equal volume (100 µL) of the standardized conidial inoculum was then added to each well. The plate was incubated at 35 °C for 48 h. The MIC was defined as the lowest essential oil concentration that resulted in the complete absence of visible fungal growth. Following the incubation period, 100 µL from each well that showed no visible growth was sub-cultured onto fresh SDA plates. The plates were incubated at 35 °C for 48 h. The MFC was defined as the lowest essential oil concentration that resulted in either no growth or fewer than three colonies on the SDA plate, corresponding to a ≥ 99.9% reduction in the original inoculum viability, indicating a fungicidal effect. Concentrations that inhibited growth in the broth but did not meet this criterion upon sub-culturing were considered fungistatic27.

Time-kill kinetics assay

The fungicidal kinetics of Syzygium aromaticum essential oil (Clove EO) and itraconazole against Aspergillus fumigatus (ATCC 13073) were evaluated using a time-kill assay according to established methodologies with modifications. A conidial suspension was prepared from a 7-day-old culture grown on SDA at 28 °C. The conidia were harvested by flooding the plate with sterile saline containing 0.05% Tween 80 and gently scraping the surface with a sterile loop. The suspension was adjusted to a final concentration of 4 × 10⁵ CFU/mL in RPMI 1640 medium buffered with 3-(N-morpholino)propanesulfonic acid (MOPS) (0.165 M, pH 7.0) using a hemocytometer for quantification. The assay was performed by incubating the conidial suspension with Clove EO and itraconazole at multiple concentrations (including 0.25x, 0.5x, x and 2x, the Minimum Inhibitory Concentration). A drug-free tube containing only the inoculum in RPMI 1640 medium served as a growth control. All tubes were incubated in a shaking incubator at 35 °C. At predetermined time intervals (0, 2, 4, 8, 12, 16, 20, and 24 h), 100 µL aliquots were removed from each tube. These aliquots were subjected to the fungicidal activity was defined as a ≥ 99.9% (or a 3-log10) reduction in the CFU/mL compared to the starting inoculum. Time-kill curves were generated by plotting the log10 CFU/mL against time28.

Animals

A total of 24 female New Zealand White rabbits, weighing 1.5–2.5 kg, were obtained. The rabbits were acclimated for at least one week prior to the commencement of the study. The room temperature was maintained at 21 ± 0.5 °C with a 12-hour light/dark cycle, and the relative humidity was set at 45%. The animals had access to water and standard rabbit chows. In the current study, all experimental procedures and methods were conducted in accordance with the guidelines of the I.R Iran Ethics Committee for Research on working with Laboratory Animals (ID: IR.UT.VETMED.REC.1401.013) and the ARRIVE guidelines.

Establishment of the non-neutropenic immunosuppressed animal model of invasive pulmonary aspergillosis

To establish the animal model, New Zealand white rabbits received cyclosporine (CsA) at a dose of 10 mg/kg/day (0.5 mg/kg during 15 s), and methylprednisolone at a dose of 5 mg/kg/day intravenously for 14 consecutive days to induce immunosuppression without neutropenia25,26,29. On the fourth day of the experiment, the fungal inoculum was delivered endotracheally under general anesthesia with ketamine and xylazine25,26,29,30.

Microorganism and conidial suspension

To prepare the inoculum, Aspergillus fumigatus strain ATCC 13,073 from a frozen isolate was subcultured onto Sabouraud dextrose agar (SDA) and incubated at 37 °C until sufficient conidiation was achieved. Subsequently, 5 mL of sterile normal saline containing 0.05% Tween 20 was added to the culture medium under a laminar flow hood. The Aspergillus fumigatus strain ATCC 13,073 grown on the medium was gently scraped using a sterile culture loop, and the resulting suspension was collected with a Pasteur pipette. The desired concentration, 1 × 108 to 1.25 × 108 conidia in a volume of 250 to 350 µL, was adjusted using a hemocytometer slide and serial dilution4,26.

Antifungal treatment

The rabbits were randomly assigned to four groups: negative control (n = 6), positive control (n = 6), treatment group A (n = 6), and treatment group B (n = 6). Antifungal treatment commenced 24 h after fungal inoculation and continued until day 12. The negative control group received normal saline at 2 mL/kg, the positive control group received itraconazole at 30 mg/kg, treatment group A received clove essential oil at 10 mg/kg, and treatment group B received clove essential oil at 20 mg/kg. All treatments were administered orally every 24 h using a syringe, slowly from the side of the mouth.

Monitoring and evaluating variables

Throughout the study, the rabbits were monitored for survival time, as well as the status and extent of lung involvement in living animals. Post-mortem evaluations included the assessment of pulmonary macroscopic lesions, pulmonary and bronchoalveolar lavage fluid (BALF) fungal burden, and the presence or absence of systemic cytotoxicity in other vital organs.

Daily monitoring

The rabbits were assessed daily for pain and welfare and were monitored for survival until day 13 post-inoculation (24 h after the final treatment). The rabbit physiognomy scale was employed to evaluate features such as the shape of the nostrils, flattening of the cheeks, position and configuration of the ears, closure of the eyes, and changes in the whiskers and sensitive facial hair. General indicators of pain and distress, including darkening of the nails and feet, pallor of the eyes, closure of the third eyelid, and failure to assume a normal posture, were also recorded.

Computing tomography (CT) scan

CT images were acquired using a 2-slice CT scanner with the protocol of a slice thickness of 1–2 mm, a pitch of 0.9, and an ultra-sharp reconstruction Kernel (U90s) in the lung window. Scans were performed on days 0, 7, and 13 post-inoculations on one rabbit from each group under general anesthesia using ketamine and xylazine29,30. The animals were positioned in sternal recumbency, and images were obtained in both sagittal and frontal planes.

Post-mortem analysis

On days 7 and 13 post-inoculation, the animals were euthanized under deep anesthesia via intravenous administration of pentobarbital sodium (60–100 mg/kg) and systematically necropsied to be used for samples and histopathological examination of the lungs as well as other vital organs30.

Macroscopic (Gross) examination

The lungs were examined for appearance changes, including hyperemia, hemorrhage, tissue consistency, and color. Other vital organs, including the heart, brain, kidneys, liver, and spleen, were inspected for indicators of systemic toxicity.

Weighing the lung tissue

The net lung weight of each rabbit was carefully measured and recorded26.

BALF fungal burden

BALF was collected from each lung for cytological examination and fungal culture. For this purpose, 10 mL of sterile normal saline was instilled into the lung via the trachea using a 12 ml sterile syringe, repeated twice. The lavage fluid was collected and centrifuged at 400 × g for 10 min. The supernatant was discarded, and 2 mL of the pellet was retained. The remaining fluid was vortexed and aliquoted into 100 µl volumes. 100 µl of the obtained liquid were mounted on a slide and examined microscopically for the presence or the absence of fungal hyphae. In addition, 100 µl of a 10⁻¹ dilution was cultured on SDA. The number of Aspergillus fumigatus colony-forming units in BALF was subsequently enumerated, and CFU/mL was calculated26.

Lung fungal burden

To assess fungal burden, a defined portion of tissue was excised from each lung lobe and weighed in the next step. The tissue was then placed in a sterile container and homogenized with sterile normal saline for 30 s. dilutions of the homogenized lung (10⁻¹ and 10⁻²) were prepared in sterile normal saline. 100 µl were then plated on SDA and incubated at 37 °C for 24 h, followed by an additional 24 h at room temperature. The number of Aspergillus fumigatus colony-forming units (CFU) in each lung lobe was subsequently enumerated, and CFU/g of tissue was calculated26.

Histopathological examination

The remaining lung tissue was fixed in 10% neutral-buffered formalin for 24–48 h for histopathological evaluation. Tissue processing was then performed using an automatic tissue processor, and paraffin-embedded tissue blocks were prepared by means of a paraffin dispenser. The tissue sections, with a thickness of 5 μm, were then stained with the routine Harris Hematoxylin-Eosin stain31. Paraffin-embedded lung samples were further subjected to Gomori methenamine silver (GMS) staining to confirm fungal infection and associated inflammatory responses. Microscopically, pulmonary lesions were assessed for the presence and extent of hemorrhage, hyperemia, necrosis, thrombosis, infiltration and accumulation of inflammatory cells in bronchi, bronchioles, and alveoli, as well as for evidence of vascular fungal invasion. Also, to investigate the composition of inflammatory cells, by using cluster of differentiation (CD) 68 and cluster of differentiation (CD) 163 markers for M1 and M2 macrophages, respectively, and myeloperoxidase (MPO) marker for neutrophils, after blocking endogenous peroxidase with H2O2 solution and antigen retrieval with sodium citrate solution at pH = 6 and by thermal method, immunohistochemistry was used32.

The stained sections were examined under a light microscope, and micrographs were captured using a camera. Quantitative analysis of inflammatory cell distribution was performed with ImageJ software. Similarly, to examine the systemic cytotoxic status, sampling of other tissue components such as heart, liver, kidney, spleen and brain was performed. After fixation in 10% neutral buffered formalin, routine hematoxylin-eosin staining slides were prepared and examined for histopathological toxicological criteria under the light microscope31. Finally, the study is in accordance with ARRIVE guidelines33.

Statistical analysis

Statistical evaluation was performed using SPSS software. To this purpose, a normality test was first conducted applying Shapiro-Wilk, and subsequently, the data were evaluated using two-way ANOVA followed by Tukey’s post-hoc test.

Results

In vitro study

GC–MS analysis

The GC-MS analysis determined the following components, including eugenol, carvacrol, trans-caryophyllene, thymol, chavicol etc. Table 1. Eugenol was the major component in studied clove essential oil with 96.81%.

Table 1 Demonstrates most components of clove essential oil used in the study.
Full size table

MIC and MFC determination

The MIC evaluation revealed, in the case of clove essential oil, the well of the microtiter plate with number 8 contains a concentration of 0.156 µg/mL with no visible turbidity (Fig. 1A). The surface-culture results of clove essential oil in concentrations of 0.156, 0.313, and 0.625 µg/mL represented no fungal growth in concentration of 0.625 µg/mL. (Fig. 1B). The MIC of Itraconazole was also determined to be 1 µg/mL, based on the well of the microtiter plate with number 5 with no visible turbidity (Fig. 1C).

Fig. 1
figure 1

(A) Clove essential oil MIC, (B) Clove essential oil MFC, (C) Itraconazole MIC.

Full size image

Time-kill kinetics

The time-kill kinetics of clove essential oil and itraconazole were evaluated against Aspergillus fumigatus at concentrations of 2×MIC, MIC, ½×MIC, and ¼×MIC, respectively, for CEO including 0.313, 0.156, 0.078, and 0.039 µg/mL, and for Itraconazole including 2, 1, 0.5, and 0.25 µg/mL. Both agents exhibited a concentration-dependent fungicidal effect. A pronounced reduction in fungal viability, achieving up to a 2-log10 decrease in colony-forming units (CFU)/mL compared to the growth control, occurred within the first 4 to 12 h of exposure.

This initial rapid kill phase was followed by a period of relative stability in the fungal burden from 12 to 24 h. Although a continued decline in CFU/mL was observed at the 2×MIC and MIC concentrations during this later phase, the reduction did not constitute an additional logarithmic kill (i.e., a further 99% reduction). These data demonstrate that the primary fungicidal activity of both clove oil and itraconazole under these conditions occurs within the first 12 h of exposure (Fig. 2).

Fig. 2
figure 2

(A) Clove essential oil time-killing curve, (B) Itraconazole time-killing curve.

Full size image

In vivo study results

CT scan

CT scans were performed on days 0, 7, and 13 post-inoculations with Aspergillus fumigatus ATCC 13,073. In the negative control group (normal saline), CT images revealed significant development and progression of nodular pulmonary lesions on days 7 and 13 compared with day 0. Between days 7 and 13, the extent of pulmonary involvement increased further. In contrast, rabbits in the positive control group (Itraconazole) exhibited fewer pulmonary lesions on day 7 post-inoculation than those in the negative control group. Following antifungal treatment, these rabbits demonstrated a favorable response to the treatment, with a reduction in patchy attenuation within the lung parenchyma between days 7 and 13. Rabbits treated with clove essential oil also showed fewer pulmonary lesions on day 7 compared with the negative control group. The higher dose (20 mg/kg) was associated with fewer lesions than the lower dose (10 mg/kg). Between days 7 and 13, lung lobe involvement in the clove-treated groups remained largely stable. Despite evident reductions in patchy attenuation and nodular pneumonia, the lesions demonstrated minimal progression, with signs of ongoing healing and a subtle decrease in lesion density. Overall, a minor difference in treatment response was observed between rabbits receiving Itraconazole and those treated with clove essential oil at 20 mg/kg on day 7. No statistically significant difference was observed between the two groups at this time point. However, by day 13 post-inoculation, the positive control group (Itraconazole) displayed a faster and better recovery (Fig. 3).

Fig. 3
figure 3

CT scans on days 0, 7, and 13 post-inoculations with Aspergillus fumigatus ATCC 13073. (A) Day 0 and before fungal inoculation, (1) negative control group, (2) positive control group (3) clove essential oil at 20 mg/kg (4) clove essential oil at 10 mg/kg (B) Day 7 post-inoculation, (1) negative control group, (2) positive control group (3) clove essential oil at 20 mg/kg (4) clove essential oil at 10 mg/kg (C) Day 13 post-inoculation (1) negative control group, (2) positive control group (3) clove essential oil at 20 mg/kg (4) clove essential oil at 10 mg/kg. The arrow shows nodular lesions involved rabbits’ lungs.

Full size image

Macroscopic examination for cytotoxicity

Macroscopic evaluation of vital organs, including the heart, kidneys, liver, spleen, and brain, following the administration of normal saline, Itraconazole, and clove essential oil at the studied doses, revealed no significant differences in lesions or signs of systemic cytotoxicity. All examined tissues exhibited normal histological structure.

Macroscopic examination of the lung

Macroscopic examination of lung tissue in rabbits from different groups on days 7 and 13 post inoculation to Aspergillus fumigatus strain ATCC 13,073, demonstrated a significant involvement of the lung lobes on day 7 in the form of small dark red hemorrhagic areas, the formation of nodules indicating an inflammatory response a variety of dark red spotted lesions, along with firm, aggregated, or confluent gray-to-white nodular lesions. Additionally, Fewer firm, flattened, confluent gray-to-red lesions were scattered throughout the lungs. On day 13 post inoculation to the fungus, nodules indicating an inflammatory response were formed. These were firm, confluent, flattened gray-to-red lesions, and less firm, aggregated, or confluent gray-to-white nodules and areas of hemorrhage. An increase was shown in the extent of pulmonary involvement in the negative control group (normal saline). In contrast, concerning the groups treated with Itraconazole and clove essential oil at doses of 20 mg/kg and 10 mg/kg, respectively, the extent of pulmonary involvement either decreased on day 13 post-inoculation or the extent of lesions did not progress.

Comparison of mean lung weights between the study groups on days 7 and 13

The results revealed a significant difference between the mean lung weights of the rabbits in the study groups, regardless of time. Tukey’s post hoc comparisons indicated that the mean lung weights in the Itraconazole group were significantly lower than those in the negative control group and the group treated with clove essential oil at a dose of 10 mg/kg (p < 0.05). However, no significant difference was observed between the mean lung weights in this group compared to the clove essential oil group at a dose of 20 mg/kg (p > 0.05). Likewise, no significant difference was observed between the mean lung weights of the other groups (p > 0.05). Regarding time, there was also no significant difference between the average lung weights of rabbits on days 7 and 13 post-inoculation, regardless of group (p > 0.05). (Table 2; Fig. 4A)

Table 2 A comprehensive summary of the statistical analyses conducted to assess lung weight in rabbits along with the quantification of fungal burden in BALF and pulmonary tissues.
Full size table

Fungal burden of BALF and lung tissue between the study groups on days 7 and 13

The analysis of the mean logarithm of fungal burden in the bronchoalveolar lavage fluid of the groups indicated a significant difference between the groups, regardless of time (p < 0.05). Tukey’s post hoc comparisons showed that the mean logarithm of BALF fungal burden in the Itraconazole group was substantially lower than that to the negative control group (p < 0.05). Still, there was no significant difference between the means of these groups compared to the other groups (p > 0.05). Furthermore, comparing the mean logarithm of BALF fungal burden at different times (irrespective of the treatment group), indicated no significant differences between days 7 and 13 post inoculation (p > 0.05). (Table 2; Fig. 4B)

Regarding the comparison of the mean logarithm of fungal burden of the rabbit lung of the studied groups, the results showed that there was a significant difference between them regardless of time (p < 0.001). Tukey’s post hoc comparisons showed that the mean logarithm of lung fungal burden in the Itraconazole group was significantly lower compared to the other groups (p < 0.05). Still, there was no significant difference between the mean logarithm of lung fungal burden in the other groups compared to each other (p > 0.05). Also, by comparing the mean logarithm of lung fungal burden at different times (regardless of group), it was observed no significant difference between days 7 and 13 post-inoculation (p > 0.05). (Table 2; Fig. 4C).

Fig. 4
figure 4

(A) Mean lung weights between the study groups on days 7 and 13 bar chart, (B) Fungal burden of BALF between the study groups on days 7 and 13 bar chart, (C) Fungal burden of lung tissue between the study groups on days 7 and 13 bar chart. Non-identical letters indicate statistically significant differences between the corresponding groups (p < 0.05).

Full size image

Histopathological examination of lung tissue

Histopathological examination of various lung lobes of the rabbits studied, including the left lung lobes (anterior and posterior) and the right lung lobes (anterior, middle, posterior and accessory) was performed at two-time intervals of 7- and 13-days post-inoculation to the Aspergillus fumigatus strain ATCC 13,073. In order to assess the extent of lung tissue involvement, inflammatory response and other pathological lesions with three criteria of severe, moderate and mild and to calculate the percentage of lung lobe involvement compared to the typical structure of rabbit lung tissue, indicated that the infiltration of inflammatory cells comprised a significant number of neutrophils (heterophiles) and a smaller number of foamy alveolar macrophages, necrosis and cellular debris (necrotizing pneumonia), focal hemorrhage, edema, fibrin deposition, Langhans giant cells, infiltration of lymphocytes and the formation of asteroid bodies to a small extent in the samples corresponding to the seventh day after exposure. Exposure to Aspergillus fumigatus strain ATCC 13,073, indicative of an acute to subacute inflammatory phase, was evident. The comparison of the above findings among the samples related to each group indicated that the highest level of involvement and pathological lung lesions was among rabbits in the negative control group without effective treatment (normal saline). In contrast, the positive control group (with itraconazole) showed the lowest level of lung involvement and pathological lesions caused by induced fungal infection and the inflammatory response associated with it. It is worth noting that in the groups treated with clove essential oil, the extent of lesions and inflammatory response in the lung tissue was lower in rabbits receiving a dose of 20 mg/kg compared to the dose of 10 mg/kg. (Fig. 5)

Fig. 5
figure 5

Macroscopic and microscopic figures of lung on 7-day post-inoculation to the Aspergillus fumigatus strain ATCC 13073. (A) Negative control group (1) Macroscopic view of a rabbit lung34, (2) Extensive pulmonary involvement (star). (3) Accumulation of proteinaceous material and edema (star). (4) Multifocal necrosis (arrowheads) and giant cell (arrow). (B) Positive control group (1) Macroscopic view of a rabbit lung (2) Significant pulmonary involvement. (3) Alveolar atelectasia and mild pulmonary involvement, H&E. (4) giant cell (arrow). (C) Clove essential oil at 20 mg/kg (1) Macroscopic view of a rabbit lung (2) Extensive pulmonary involvement along with hemorrhage (star). (3) Accumulation of proteinaceous material and edema (star). (4) focal necrosis (arrowhead) and giant cell (arrow). (D) Clove essential oil at 10 mg/kg (1) Macroscopic view of a rabbit lung (2) Extensive pulmonary involvement. (3) Multifocal necrosis (arrowheads). (4) giant cell (arrow), H&E.

Full size image

In the examination of samples from day 13 after exposure to Aspergillus fumigatus strain ATCC 13,073, a shift in the proportion of inflammatory cells was observed with an increase in the number of macrophages along with the formation of Langhans giant cells and numerous foreign bodies, infiltration of neutrophils, lymphoplasmacytic bronchiolitis, necrotic areas and cellular debris, hyperplasia of type II pneumocytes and the formation of abundant asteroid bodies consisting of small hyphae and ungerminated conidia of the fungus surrounded by inflammatory cells of neutrophils and epithelioid macrophages and deposition of amorphous eosinophilic protein materials in a radial and spheroidal arrangement indicating the onset of a chronic inflammatory response and a pyogranulomatous inflammatory reaction. In comparison between the studied groups, lung lesions and inflammatory response in rabbits in the negative control group without effective treatment (normal saline) showed the highest levels, and with time, the progression of pathological lesions due to the inflammatory response to the induced fungal infection was evident. The positive control group (with Itraconazole) had the lowest involvement in lung tissue, and with time and drug administration, the severity of lung lesions decreased from day 7 to 13. In the groups receiving clove essential oil, the involvement of lung tissue at a dose of 20 mg/kg was generally less than that at a dose of 10 mg/kg, and the effects of improvement and reduction of inflammatory response from day 7 to 13 at a dose of 20 mg/kg were significantly greater than those at a dose of 10 mg/kg. (Fig. 6)

Fig. 6
figure 6

Macroscopic and microscopic Figures of lung on 13-day post-inoculation to the Aspergillus fumigatus strain ATCC 13073. (A) Negative control group (1) Macroscopic view of rabbit lung34, (2) Extensive pulmonary involvement along with pyogranulomas (arrowhead). (3) Accumulation of neutrophils within alveolar spaces (arrowheads). (4) Multifocal Splendore-Hoeppli phenomenon formation (arrow), H&E. (B) Positive control group (1) Macroscopic view of rabbit lung (2) Focal pulmonary involvement (star). (3) Focal pulmonary involvement and giant cell (arrowhead), H&E. (4) Multifocal Splendore-Hoeppli phenomenon formation (arrow). (C) Clove essential oil at 20 mg/kg (1) Macroscopic view of rabbit lung (2) Focal pulmonary involvement (star). (3) Focal pulmonary involvement and Splendore-Hoeppli phenomenon formation (arrow). (4) Focal Splendore-Hoeppli phenomenon formation (arrow). (D) Clove essential oil at 10 mg/kg (1) Macroscopic view of rabbit lung (2) Extensive pulmonary involvement accompanied by perivascular inflammation (arrowheads). (3) Alveolar atelectasis along with giant cell (arrowhead). (4) Numerous giant cell (arrows), H&E.

Full size image

Histochemistry and immunohistochemistry examination of lungs

The presence of fungal structures scattered within rabbits’ lungs and related inflammatory responses was confirmed by using GMS staining. Examination of the expression of the CD68 marker associated with M1 macrophages in lung samples on day 13 after exposure to Aspergillus fumigatus strain ATCC 13,073 in the studied groups showed that the normal saline group had higher expressions compared to other groups due to the severe inflammatory response to the fungus. After that, the clove essential oil group at a dose of 10 mg/kg had significant marker expression, while the itraconazole and clove essential oil groups at a dose of 20 mg/kg had the lowest levels, respectively.

Examination of the expression of the CD163 marker associated with M2 macrophages in lung samples on day 13 after exposure to Aspergillus fumigatus strain ATCC 13,073 in the studied groups showed that in the normal saline group, due to greater fungal contamination and the effect of Aspergillus fumigatus on the differentiation of M1 to M2 macrophages, the expression of the marker was higher than in the other groups, and in the itraconazole and clove essential oil groups at doses of 20 mg/kg and 10 mg/kg, respectively, it was lower.

Examination of the expression of MPO marker as an indicator for neutrophil differentiation in lung samples on day 13 after exposure to Aspergillus fumigatus strain ATCC 13,073 in the studied groups showed that the marker expression was lower in the itraconazole group as well as the clove essential oil group at a dose of 20 mg/kg than in the other groups. However, no notable changes were observed between the normal saline and clove essential oil groups at a dose of 10 mg/kg (Fig. 7).

Fig. 7
figure 7

Histochemistry and Immunohistochemistry examination of rabbits’ lungs on 13-day post-inoculation to the Aspergillus fumigatus strain ATCC 13073. (A) Negative control group (1) The arrow shows fungal structure, GMS staining (2) Extensive expression of CD68. (3) Remarkable expression of CD163. (4) Extensive expression of MPO. (B) Positive control group (1) The arrow shows fungal structure, GMS staining (2) Low expression of CD68. (3) Insignificant expression of CD163. (4) Mild expression of MPO. (C) Clove essential oil at 20 mg/kg (1) The arrow shows fungal structure, GMS staining. (2) Low expression of CD68. (3) Insignificant expression of CD163. (4) Mild expression of MPO. (D) Clove essential oil at 10 mg/kg (1) The arrow shows fungal structure, GMS staining (2) Significant expression of CD68. (3) Moderate expression of CD163. (4) Relatively notable expression of MPO.

Full size image

Comparison of lung lesions between the study groups on days 7 and 13

The extent of lung involvement as determined by histopathology and macrophages type 1 and 2 and neutrophils specific markers in the control and clove essential oil-treated groups on days 7 and 13 post inoculation has been summarized as follows. Overall, Tukey’s post-hoc comparisons revealed that pulmonary lesions in rabbits being treated with Itraconazole were significantly less compared to those in the negative control group (without effective treatment) and the clove essential oil-treated group at a dose of 10 mg/kg (p < 0.05). However, no significant differences were observed in the pulmonary lesions of rabbits in this group compared to the clove essential oil-treated group at a dose of 20 mg/kg (p > 0.05). Additionally, no significant differences were detected between the pulmonary lesions in other groups (p > 0.05). Regarding time, there was no significant difference between the pulmonary lesions of rabbits on days 7 and 13 post inoculation, regardless of treatment group (p > 0.05) (Table 3; Fig. 8).

Table 3 An analytical overview of the statistical evaluations undertaken to assess IHC markers expression and to elucidate the degree of pulmonary involvement in the rabbit model.
Full size table
Fig. 8
figure 8

(A) Percentage of CD68 expression bar chart, (B) Percentage of CD163 expression bar chart, (C) Percentage of MPO expression bar chart, (D) Percentage of pulmonary involvement bar chart. Non-identical letters indicate statistically significant differences between the corresponding groups (p < 0.05).

Full size image

Microscopic examination of other tissue components

Microscopic examination of the heart, kidney, liver, spleen, and brain of rabbits treated with clove essential oil at the studied doses (including two doses of 10 and 20 mg/kg), normal saline and Itraconazole treated groups on days 7 and 13 post-Inoculation, revealed ordinary histologic appearance. No evidence of systemic cytotoxicity including hemorrhage and necrosis was observed and recorded in any group (Figs. 9 and 10).

Fig. 9
figure 9

Histopathological examination of vital organs after receiving treatments for 6 days. H&E. (A) Heart (1) Negative control group (2) Positive control group (3) Clove essential oil at 20 mg/kg (4) Clove essential oil at 10 mg/kg. (B) Kidney (1) Negative control group (2) Positive control group (3) Clove essential oil at 20 mg/kg (4) Clove essential oil at 10 mg/kg. (C) Liver (1) Negative control group (2) Positive control group (3) Clove essential oil at 20 mg/kg (4) Clove essential oil at 10 mg/kg. (D) Spleen (1) Negative control group (2) Positive control group (3) Clove essential oil at 20 mg/kg (4) Clove essential oil at 10 mg/kg. E) Brain (1) Negative control group (2) Positive control group (3) Clove essential oil at 20 mg/kg (4) Clove essential oil at 10 mg/kg, H&E.

Full size image
Fig. 10
figure 10

Histopathological examination of vital organs after receiving treatments for 12 days. H&E. (A) Heart (1) Negative control group (2) Positive control group (3) Clove essential oil at 20 mg/kg (4) Clove essential oil at 10 mg/kg. (B) Kidney (1) Negative control group (2) Positive control group (3) Clove essential oil at 20 mg/kg (4) Clove essential oil at 10 mg/kg. (C) Liver (1) Negative control group (2) Positive control group (3) Clove essential oil at 20 mg/kg (4) Clove essential oil at 10 mg/kg. (D) Spleen (1) Negative control group (normal saline) (2) Positive control group (3) Clove essential oil at 20 mg/kg (4) Clove essential oil at 10 mg/kg. (E) Brain (1) Negative control group (2) Positive control group (3) Clove essential oil at 20 mg/kg (4) Clove essential oil at 10 mg/kg, H&E.

Full size image

Discussion

In the current study, clove essential oil, with eugenol being the main active ingredient, exhibited a high volume of 96.81% which exceeded all the previously reported studies (85–90%)14,19. Due to the numerous properties of plant compounds, particularly clove plant, in destroying various microorganisms such as bacteria and fungi35, as well as considering the increased resistance of Aspergillus fumigatus to azole antifungal drugs following some mutations such as TR34/L98H and TR46/Y121F/T289A36, extensive studies have been conducted to confirm the significant effects of minimal concentrations of plant compounds on bactericidal and fungicidal activity, particularly on Aspergillus fumigatus27. Both pure and nanoemulsion forms of clove essential oil have revealed significant bactericidal properties20. In the current study, MFC and MIC of clove essential oil as an herbal compound with a high percentage of eugenol showed satisfactory efficiency against Aspergillus fumigatus strain ATCC 13,073 comparable to Itraconazole as a commercial antifungal drug. For which the MIC for Itraconazole was calculated to be 1 µg/mL. This was notable as in the previous studies conducted on isolated Aspergillus fumigatus isolates with the TR34/L98H mutation, all had MICs greater than 1 µg/mL for Itraconazole and 97% had MICs greater than 1 µg/mL and more than 0.25 µg/mL for Voriconazole and Posaconazole, respectively. Also, all the isolated Aspergillus fumigatus isolates with the TR46/Y121F/T289A mutation had MICs greater than 1 µg/mL for Voriconazole, 76% had MICs greater than 1 µg/mL and 35% had MICs greater than 0.25 µg/mL for Itraconazole and Posaconazole, respectively36. Examination of the time-killing assay curve of Aspergillus fumigatus strain ATCC 13,073 for diverse concentrations of clove essential oil represented a similar downward trend in fungal burden and decrease (2Log) from 4 to 12 h along with an insignificant decline from 12 to 24 h after inoculation compared to the control sample in comparison with Itraconazole different concentrations. Similar evaluations were also reported in a study using tea tree essential oil. The pure concentration of the essential oil showed the most significant and fastest impacts with a decline of about 4Log from the initial hours of exposure (4 h), and the dilutions showed a slower downward trend28. In another study in also found that clove essential oil was able to show effective performance in controlling fungal growth and mycotoxin production in different strains of Aspergillus37. The fungicidal and bactericidal effects of clove essential oil at various concentrations against Candida albicans, Escherichia coli, and Staphylococcus aureus had also been confirmed in previous studies38.

In addition to extensive, in vitro investigations have been conducted on Aspergillus spp., in vivo studies utilizing animal models have been increased15. Also, today, antifungal treatment recommendations have been updated in combination of first-line and second-line medicines. Meanwhile, increasing attention has been paid to the use of new methods and combinations16.

A very high mortality rate was observed in the method of creating a pulmonary aspergillosis model with immunosuppression and persistent neutropenia in the groups without effective treatment24,25,26, whereas in non-neutropenic immunosuppression method, the mortality rate of rabbits of negative control group reached zero.

In addition to finding new antifungal agents, concerns regarding to establishing a rapid and definitive method for diagnosing invasive pulmonary aspergillosis and achieving a less invasive procedure for initiating antifungal treatments have caused diverse techniques such as molecular imaging and PET by using animal models have been evaluated39,40. In other similar studies, conducted on New Zealand White rabbits and using Aspergillus spp. such as A. fumigatus and A. tereus, CT scan images played an important role as a diagnostic method at different time points29,41. CT scans have also been recognized in non-neutropenic patients with nodular lesions as a diagnostic method for pulmonary aspergillosis1,42.

In the current study, rabbits’ CT scans have exhibited the relatively healthy lung appearance change to the involvement of various lobes after endotracheal conidial suspension inoculation. Its extension was noticeable in the negative control group. Nevertheless, in other groups treated with Itraconazole and clove essential oil, respectively, at a dose of 20 mg/kg and 10 mg/kg, less pulmonary lesions and inflammatory response, and following less nodular pulmonary involvement were evident due to their antifungal effects. After 13 days of inoculation, a slight upward trend in pulmonary lesions was observed in the negative control group, indicating the lack of effective treatment administration in this group, whereas a reduction in the pulmonary involvement and lesions indicating the significant effectiveness of the treatment in the Itraconazole group was observed. However, the group clove essential oil at dose 20 mg/kg was relatively similar at the Itraconazole group at day 7 posy-inoculation, the changes from day 7 to day 13 were insignificant in groups treated with clove essential oil. However, the lack of progression of the inflammatory response accompanied by a gentle healing process in the lung tissue at the time of using clove essential oil has been noticed.

No Aspergillus fumigatus hyphae were observed in the examination of bronchoalveolar lavage fluid under a light microscope, which is due to the type of immunosuppression without neutropenia, where the presence of neutrophils prevented the creation of abundant fungal hyphae compared to the immunosuppression method with continuous neutropenia5,26,29.

The lung weight mean analysis revealed that positive control group with Itraconazole treatment had a lower mean weight, and it decreased with continued treatment. In the clove essential oil treatment groups, the mean lung weight in the group with a dose of 20 mg/kg and 10 mg/kg was respectively less than the negative control group without effective treatment and greater than the positive control group receiving Itraconazole. This reduction in mean lung weight was also evident on days 7 to 13. In other studies, conducted using immunosuppressed rabbits with persistent neutropenia and without neutropenia, a similar condition was observed in which a rise in lung weight mean among control groups without effective treatment up to more than 45 g and up to 20 g in samples treated with commercial drugs during the experiment was evident4,24,26,41.

Similar to lung weight mean, generally BALF and lung lobes fungal burden in the group without effective treatment recorded the highest and had an upward trend from day 7 to 13 post-inoculation. In contrast, the lowest fungal burden belonged to the group with Itraconazole treatment which decreased from day 7 to 13 post-inoculation consistently. The groups treated with clove essential oil placed between these two controlled groups, and the group treated with a dose of 20 mg/kg indicated more efficient fungal burden reduction. In a study with inducing pulmonary infection in BALB/c mice using intratracheal administration of lipopolysaccharide, the aqueous extract of clove plant administered intraperitoneally reduced the number of neutrophils and protein leakage into the bronchoalveolar lavage fluid significantly. In addition, in vitro study has exhibited positive effects of clove on reducing MPO activity43. In other similar studies, the control group without effective treatment had the highest fungal burden and the other groups treated with different agents had a lower fungal burden4,26,41.

Vital organs’ examination in two ways of macroscopic and microscopic showed no signs of cellular and systemic toxicity due to using clove essential oil compared to the control groups. In the other studies conducted, no evidence of cytotoxicity has been reported so far35,44. In addition, the absence of subchronic toxicity and mutagenicity has been confirmed concerning the polyphenolic extract of clove seeds45.

In a similar attitude to using herbal compounds, extensive studies exist one of which has revealed the active ingredient cinnamaldehyde, obtained from the essential oil of cinnamon from the Lauraceae family, can have a significant antifungal effect in an animal model of invasive pulmonary aspergillosis in immunosuppressed ICR mice46. Another study, however, revealed a significant effect in eliminating Aspergillus and reducing damage to lung tissue silver nanoparticles from the essential oil of Artemisia sieberi in Swiss albino mice after inducing immunosuppression with neutropenia and infection with the fungus Aspergillus fumigatus by intranasal administration47. In another similar study, the antifungal activity of gold nanoparticles of Leptadena hastata essential oil was investigated and confirmed in Swiss albino mice48. Another example of these studies is the demonstration of the antifungal effect of Calotropis gigantean extract in a model of invasive pulmonary aspergillosis in neutropenic mice49. Not only have herbal agents’ efficiency in antifungal therapy been evaluated, but also their other potentials particularly about clove plant have been of great interests. So, in a study, it was found that the use of clove essential oil encapsulated by nanofibers shows sound effects on wound healing and antimicrobial activity against Escherichia coli and Staphylococcus aureus50. In addition to its bactericidal properties, eugenol has been shown having properties in eliminating parasites such as Giardia lamblia, Fasciola gigantica, Haemonchus contortus, and Schistosoma mansoni51. In this regard, the relative effectiveness of clove essential oil in improving liver lesions was observed in a study which was conducted using various plant essential oils, including clove essential oil, on rabbits infected with Eimeria estidae52. In another study using cold-pressed clove oil, its protective properties against liver cells being damaged by CCL4 were noted, which may indicate that clove products, in addition to not having toxic effects and causing toxicity lesions, have significant effectiveness in improving hepatotoxic lesions induced, probably due to antioxidant compounds53. In a study in 2016, the protective effect of clove powder was also evident in rabbits, as in the group that first received paracetamol, liver enzymes including ALT, AST, GGT, ALP and LDH substantially increased after receiving clove powder, and decreased after administration. No changes were observed in the group that received it simultaneously54. In a study on rats in which diabetes mellitus was induced, the anti-hyperglycemic effects of aqueous extracts of various plants, including Syzgium cumini (Jamun), were evaluated. The results of this study confirmed the reducing effect on blood sugar levels and weight changes in diabetic rats55. In another study, clove essential oil emulsion showed significant antidiabetic properties using alpha-amylase and starch enzymes56. In a study, in rats been induced with oxidative stress by H2O2, administration of clove essential oil resulted in positive effects in brain, liver, and kidney tissues, as well as blood biochemical indicators57. Clove essential oil is also known as an antioxidant compound in food and pharmaceutical compositions by increasing the shelf life and maintaining their nutritional properties58. In a study, it was found that low doses of clove essential oil could be effective in improving and controlling oxidative stress, apoptosis, and excessive autophagy59. In addition, in another study, it was found that short-term consumption of clove essential oil can have antidepressant properties and long-term consumption has anti-stress properties by improving the hippocampal pERK1/2-pCREB-BDNF pathway in rats60. Likewise, various studies approved that the eugenol antifungal activity operates via several mechanisms, predominantly by targeting the cell membrane of fungi. Eugenol has been demonstrated to interact with fungal membranes and suppress biosynthesis of ergostrol that is an essential component in providing function and integrity of membrane. Consequently, it can result in destabilization of cell membrane and enhanced permeability, ultimately causing leakage of intracellular contents and aberrant cell morphology61,62.

In current study, in the macroscopic and microscopic examination of lung tissue, pulmonary lesions in the negative control group were much more severe compared with the other groups accompanied by an upward trend from day 7 to 13 post-inoculation. Conversely, the positive control group being treated with Itraconazole showed the mildest pulmonary lesions with a declining and recovery tendency from day 7 to 13 post-inoculation. The groups treated with clove essential oil the extent and following reduction of pulmonary lesions was generally slightly greater in the group treated with a dose of 20 mg/kg.

Comparison of histopathological findings of the current study with others represented similar pulmonary lesions. As in studies by using immunosuppressed rabbits with both non-neutropenic and persistent neutropenia and treated with the commercial antifungal drugs such as amphotericin B, extensive fungal hyphal formation and extensive tissue necrosis with vascular infiltration and extensive hemorrhage and vascular thrombosis without significant inflammatory cell infiltration were observed in the persistent neutropenic groups compared to untreated control rabbits without neutropenia. However, in the non-neutropenic groups, the predominant lesions were inflammatory cell accumulation in the form of granulomas containing neutrophils and macrophages, necrosis and without extensive fungal hyphal formation. In the treated rabbits, the group of immunosuppressed rabbits without neutropenia also showed a better condition24,25,26.

In addition, the type M1 macrophages’ ratio in the negative control group was higher than in the other groups, which could be due to the high level of fungal infection and burden. The population of M2 macrophages was also higher in the negative control group than in the other groups due to the effect of the fungus regarding higher burden. It should be noted that it has been shown that Aspergillus fumigatus can cause a change in the phenotype of activated macrophages to M2 type by upregulating M2 macrophage markers such as transcripts of the gene encoding arginase-1 (Arg1), Ym1, and CD2065. This was consistent with the control group without effective treatment. In the treated groups, itraconazole, clove essential oil 20 mg/kg and 10 mg/kg groups were in better condition and antigen expression was lower, respectively. The expression of neutrophil-specific marker was relatively analogous related to extension of pulmonary involvement and fungal burden in diverse groups. It should be noted that the effect of clove essential oil in regulating the immune response has been investigated and confirmed in both the humoral and cell-mediated immune systems63. It was also determined that the components of clove can suppress the release of interferon gamma, and induce the secretion of interleukins 4 and 10, as well as TGF-B and as a result can suppress T lymphocyte cellular immunity and increase humoral immune responses, so that the cytokine pattern changes towards Th2 and regulatory responses and increases humoral immune cytokines64.

Notwithstanding most pathological findings has been related to experimental studies, a case report study has represented the pulmonary lesions including scattered nodular lesions throughout the lungs on macroscopic examination and the presence of multiple pyogranulomatous lesions with the formation of asteroid bodies, infiltration of neutrophils, epithelioid macrophages, and the formation of multinucleated giant cells on microscopic examination of apparently healthy rabbits which infected by Aspergillus65.

This study faced several methodological limitations that should be acknowledged. First, because our country in an importer of certain specialized pharmaceuticals-including cyclosporine-access of this drug for research purposes was limited and difficult. Consequently, the minimum feasible number of animals was allocated to each experimental group based on the restricted amount of cyclosporin available. Second, although the investigation focused on pathological alterations across all pulmonary lobes, only a small and standardized portion of each lobe was sampled for fungal burden quantification. Individual variations among rabbits during anesthesia and differences in respiratory rhythm resulted in slight inconsistencies in fungal distribution within lung tissue, leading to a higher-than-expected deviation in fungal burden measurements. Finally, due to the limited availability of IHC antibodies raised against animal-derived antigens in our setting, human-specific antibodies were employed. These antibodies were carefully evaluated and optimized using rabbit positive-control tissues prior to use, however, this substitution may have introduced minor technical variability.

Conclusion

Summarily, the current study represented that clove essential oil had a significant and important effectiveness in inhibiting the growth and even eliminating Aspergillus fumigatus strain ATCC 13,073, as a filamentous fungus, compared to Itraconazole, as a commercial medicine, from the azole family in in vitro experiment. In in vivo experiments, the findings related to CT scan images, lung tissue weight, bronchoalveolar lavage fluid and lung tissue fungal burden, and macroscopic and microscopic lung examination were very consistent, and an increasing trend was observed in the negative control group without receiving effective treatment, and a decreasing trend and improvement were observed in the groups treated with Itraconazole and different doses of clove essential oil in two time periods of 7 and 13 days post-inoculation to Aspergillus fumigatus strain ATCC 13,073, even though the changes sometimes were slight and differences might be scant between them. It can also be noted that the findings of the current study were consistent with most other studies, both in vitro and in vivo. An Essential point in this study was that although Itraconazole showed more significant results in terms of reducing the fungal burden in the tissue and reducing the extent of pulmonary involvement and lesions than other groups, clove essential oil without causing symptoms of systemic toxicity at a higher dose exhibited relatively better performance compared to a lower dose. Both groups treated with clove essential oil showed effective results compared to the negative control group without receiving effective treatment. This could suggest that if clove essential oil were administered at higher doses, different time intervals, or different formulations, better results might be observed in reducing the fungal burden, inflammatory response, and pulmonary lesions.

The following are suggestions for conducting additional studies in this field, such as using other forms of clove essential oil to improve absorption and performance, such as Nano-compounds and emulsions, increasing the drug dose or administration intervals, using a combination of a commercial drug and clove essential oil, comparing the performance of clove essential oil in reducing fungal lesions in two different groups of filamentous and yeast fungi, and comparative assessment of pure molecules with extracts for their antifungal potential.