Antifungal susceptibility pattern of Aspergillus spp. isolated from dogs and cats with mycotic dermatitis

Introduction

Mycotic dermatitis is an important fungal skin disorder affecting dogs and cats and is commonly associated with alopecia, erythema, scaling, crust formation and pruritus. The condition is caused by a variety of fungal pathogens and opportunistic fungi, leading to considerable clinical importance in companion animal practice (Moriello, 2014; Cafarchia et al., 2006) [1,2] . Recent advances in veterinary mycology have indicated an increasing occurrence of fungal dermatitis associated with opportunistic non-dermatophytic fungi, among which Aspergillus spp. are considered predominant due to their frequent isolation from dermatological and superficial lesions in companion animals (Tell, 2005; Segal, 2009) [3,4] . Aspergillus spp. are ubiquitous environmental fungi commonly present in soil, organic debris and air. Species such as A. niger, A. flavus and A. fumigatus have been associated with superficial, cutaneous and systemic infections in animals, particularly in immunocompromised animals or animals with impaired skin barriers (Quinn et al., 2011; Muller et al., 2013) [5,6] . The identification of Aspergillus spp. associated with dermatological lesions is important due to their pathogenic potential and variable response to antifungal therapy. In recent years, emerging antifungal resistance among opportunistic fungal pathogens has become a growing concern in both veterinary and human medicine, thereby necessitating antifungal susceptibility testing for better therapeutic management (Verweij et al., 2016; Chowdhary et al., 2017) [7,8] . Therefore, the present study was undertaken to isolate and identify Aspergillus spp. associated with fungal dermatitis in dogs and cats and to determine their in vitro antifungal susceptibility pattern against commonly used antifungal agents.

Materials and Methods

Sample collection

The present study was conducted at NTR College of Veterinary Science, Gannavaram (AP, India) during the period from August 2025 to January 2026. Clinical samples were collected from dogs and cats presented with dermatological manifestations such as alopecia, scaling, crust formation, erythema, malodour and pruritic skin lesions suggestive of mycotic dermatitis. Samples comprising skin scrapings and hair materials were collected aseptically from the active lesions using sterile scalpel blades after cleansing the affected area with 70% alcohol. The collected samples were transferred into sterile containers, properly labelled and transported to the laboratory for mycological examination.

Isolation and identification of Aspergillus spp.

The collected clinical samples were inoculated onto Sabouraud's Dextrose Agar (SDA) supplemented with chloramphenicol and cycloheximide for selective fungal isolation (Larone, 2011) [9] . The inoculated media were incubated aerobically at 28 °C and monitored periodically for fungal growth (Quinn et al., 2011). Fungal colonies exhibiting visible growth within 2 days to a week of incubation were subcultured onto fresh SDA plates to obtain pure isolates. Macroscopic colony characteristics including colony colour, texture, surface topography and reverse pigmentation were recorded following standard mycological procedures (Samson et al., 2014) [10] . Microscopic examination was performed by lactophenol cotton blue (LPCB) staining technique by placing a drop of LPCB stain on a clean glass slide followed by teasing a small portion of fungal material using a sterile needle (Benson, 2001) [11] . The stained fungal preparations were examined microscopically for the presence and arrangement of septate hyphae, conidiophores, vesicles, phialides and conidia for fungal identification (Klich, 2002; Geiser et al., 2007) [12,13] .

Antifungal Susceptibility Testing

Antifungal susceptibility testing of the fungal isolates was performed by agar based disc diffusion method using modified Mueller-Hinton agar supplemented with 2% glucose and 0.5 µg/ml methylene blue dye (Espinel-Ingroff et al., 2007; CLSI, 2017) [14,15] . The fungal inoculum was standardized visually by comparison with 0.5 McFarland turbidity standard prior to inoculation. The standardized fungal inoculum was evenly inoculated onto the surface of modified Mueller-Hinton agar plates using sterile cotton swab or sterile L-shaped spreader and antifungal discs were placed aseptically over the media. The antifungal agents used in the study were acquired from HiMedia, India included itraconazole (10 µg), ketoconazole (30 µg), fluconazole (10 µg), miconazole (30 µg), nystatin (50 µg), clotrimazole (10 µg) and amphotericin B (20 µg). The inoculated plates were incubated at 28 °C and examined for zone of inhibition from 2 days up to 1 week of incubation. The antifungal susceptibility pattern of the isolates was determined based on the zone of inhibition around the antifungal discs (Pfaller et al., 2004) [16] .

Results and Discussion

A total of 29 isolates of Aspergillus spp. were recovered from dogs and cats affected with mycotic dermatitis. The fungal isolates exhibited characteristic colony morphology on SDA and microscopic examination revealed septate hyphae, conidiophores, vesicles, phialides and conidia characteristic of Aspergillus spp. (Fig. 1 and2). Based on colony characteristics and microscopic morphology, further species level identification was made revealing 12 isolates of A. niger, 10 isolates of A. fumigatus and 7 isolates of A. flavus. On the obverse side, all isolates initially appeared as white colonies which gradually developed characteristic pigmentation with maturation. Isolates of A. niger exhibited black granular colonies, whereas A. flavus isolates showed yellowish-green colonies with powdery texture. Isolates of A. fumigatus exhibited olive-green colonies with velvety texture and a white periphery. On the reverse side, all isolates of Aspergillus spp. exhibited pale to colourless pigmentation. The cultural characteristics observed in the present study were in accordance with the standard morphological descriptions of Aspergillus spp. reported by Klich (2002) [12] , Quinn et al.

(2011) [5] and Samson et al. (2014) [10] . Similar observations on colony pigmentation, conidial arrangement and microscopic morphology of Aspergillus spp. from clinical and environmental sources have also been documented previously by Geiser et al. (2007) [13] . Isolation of A. niger, A. fumigatus and A. flavus from dermatological lesions in companion animals observed in the present investigation corroborates earlier reports describing these Aspergillus spp as opportunistic fungal pathogens associated with superficial and cutaneous infections in animals (Tell, 2005; Segal, 2009) [3,4] . Antifungal susceptibility testing of isolates of Aspergillus spp. against seven antifungal agents revealed varying zone of inhibition values among the isolates tested (Table 1). Fluconazole exhibited the lowest zone of inhibition in all the isolates of Aspergillus spp. tested, indicating comparatively lower in vitro activity against the Aspergillus isolates recovered in the present study. Similar reduced susceptibility and variable antifungal response among isolates of Aspergillus spp. towards azole antifungal agents have been documented previously by Escribano et al. (2012) [17] , Heo et al. (2015) [18] , Verweij et al. (2016) [7] and Chowdhary et al.

(2017) [8] . In A. niger isolates, ketoconazole and miconazole recorded the highest mean zone of inhibition values, whereas amphotericin B and ketoconazole exhibited comparatively higher zone diameters against A. flavus isolates. Similarly, A. fumigatus isolates demonstrated higher zone of inhibition values towards amphotericin B, ketoconazole and miconazole.

Comparable findings on the in vitro activity of amphotericin B and azole antifungal agents against isolates of Aspergillus spp. have also been reported by Herbrecht et al. (2002) [19] , Gupta et al. (2015) [20] and Vahedi-Shahandashti et al. (2023) [21] . Variability in antifungal susceptibility patterns observed among isolates of Aspergillus spp. in the present investigation may be attributed to species variation and intrinsic differences in antifungal susceptibility among opportunistic moulds. Similar observations regarding variation in antifungal susceptibility testing methods, antifungal response and interpretive criteria among Aspergillus isolates have been discussed previously by Rex et al. (2001) [22] , CLSI (2013) [23] , Berkow et al. (2020) [24] and Arendrup et al. (2023) [25] . The findings of the present study emphasize the importance of antifungal susceptibility testing for selection of appropriate antifungal agents in the therapeutic management of Aspergillus-associated mycotic dermatitis in companion animals.

Acknowledgement

AcknowledgementThe authors are thankful to the Department of Veterinary Microbiology, NTR College of Veterinary Science, Gannavaram, Sri Venkateswara Veterinary University (SVVU), Andhra Pradesh, for providing the necessary facilities and financial support to carry out this research work.

References

1. Moriello KA. Diagnostic techniques for dermatophytosis in dogs and cats. Clin Dermatol. 2014;32(2):244-248.
2. Cafarchia C, Romito D, Capelli G, Guillot J, Otranto D. Isolation of Microsporum canis from the hair coat of pet dogs and cats belonging to owners diagnosed with M. canis tinea corporis. Vet Dermatol. 2006;17:327-331.
3. Tell LA. Aspergillosis in mammals and birds: impact on veterinary medicine. Med Mycol. 2005;43 Suppl 1.
4. Quinn PJ, Markey BK, Leonard FC, FitzPatrick ES, Fanning S, Hartigan PJ. Veterinary Microbiology and Microbial Disease. 2nd ed. Oxford: Wiley-Blackwell; 2011.
5. Muller GH, Kirk RW, Scott DW, Miller WH, Griffin CE. Muller and Kirk's Small Animal Dermatology. 7th ed. Philadelphia: Saunders Elsevier; 2013.
6. Verweij PE, Chowdhary A, Melchers WJG, Meis JF. Azole resistance in Aspergillus fumigatus: can we retain the clinical use of mold-active antifungal azoles? Clin Infect Dis. 2016;62(3):362-368.
7. Chowdhary A, Sharma C, Meis JF. Azole-resistant aspergillosis: epidemiology, molecular mechanisms and treatment. J Infect Dis. 2017;216 Suppl 3.
8. Larone DH. Medically Important Fungi: A Guide to Identification. 5th ed. Washington (DC): ASM Press; 2011.
9. Samson RA, Visagie CM, Houbraken J, Hong SB, Hubka V, Klaassen CH, et al. Phylogeny, identification and nomenclature of the genus Aspergillus. Stud Mycol. 2014;78:141-173.
10. Benson HJ. Microbiological Applications: Laboratory Manual in General Microbiology. 8th ed. New York: McGraw-Hill; 2001.
11. Klich MA. Identification of Common Aspergillus Species. Utrecht: Centraalbureau voor Schimmelcultures; 2002.
12. Geiser DM, Klich MA, Frisvad JC, Peterson SW, Varga J, Samson RA. The current status of species recognition and identification in Aspergillus. Stud Mycol. 2007;59:110.
13. Espinel-Ingroff A, Arthington-Skaggs B, Iqbal N, Ellis D, Pfaller M, Messer S, et al. Multicenter evaluation of a standardized method for antifungal disk diffusion susceptibility testing of filamentous fungi. J Clin Microbiol. 2007;45(12):4038-4041.
14. Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard. 2nd ed. CLSI document M38-A2. Wayne (PA): CLSI; 2017.
15. Pfaller MA, Messer SA, Boyken L, Tendolkar S, Hollis RJ, Diekema DJ. In vitro activities of voriconazole, posaconazole and four licensed systemic antifungal agents against clinical isolates of Aspergillus species. J Clin Microbiol. 2003;41(3):1186-1189.
16. Escribano P, Peláez T, Muñoz P, Bouza E, Guinea J. Is azole resistance in Aspergillus fumigatus a problem in Spain? Antimicrob Agents Chemother. 2013;57(6):28152820.
17. Heo MS, Shin JH, Choi MJ, Park YJ, Lee HS, Koo SH, et al. Resistance to azole antifungal agents in Aspergillus isolates from Korea. J Clin Microbiol. 2015;53(2):606608.
18. Herbrecht R, Denning DW, Patterson TF, Bennett JE, Greene RE, Oestmann JW, et al. Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis. N Engl J Med. 2002;347(6):408-415.
19. Gupta AK, Mays RR, Versteeg SG, Piraccini BM, Shear NH, Piguet V, et al. Update on current approaches to diagnosis and treatment of onychomycosis. Expert Rev Anti Infect Ther. 2015;13(8):929-938.
20. Vahedi-Shahandashti R, Lass-Flörl C. Novel antifungal agents and their activity against Aspergillus species. J Fungi. 2020;6(4):213.
21. Rex JH, Pfaller MA, Walsh TJ, Chaturvedi V, EspinelIngroff A, Ghannoum MA, et al. Antifungal susceptibility testing: practical aspects and current challenges. Clin Microbiol Rev. 2001;14(4):643-658.
22. Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. CLSI document M38-A2. Wayne (PA): CLSI; 2013.
23. Berkow EL, Lockhart SR, Ostrosky-Zeichner L. Antifungal susceptibility testing: current approaches and future directions. J Fungi. 2020;6(3):1-16.
24. Arendrup MC, Meletiadis J, Mouton J, Guinea J, CuencaEstrella M, Lagrou K, et al. EUCAST technical note on isavuconazole breakpoints for Aspergillus, itraconazole breakpoints for Candida and updates for the antifungal susceptibility testing method documents. Clin Microbiol Infect. 2016;22(6):571.e1-571.e4.