Antifungal sensitivity testing of Aspergillus species isolated from

recurrent canine skin infection.

Rinmuanpuii Ralte*, S.N.S. Randhawa, Vipan Kumar., Andleeb Malik., M.Y Wani and
Naripjeet Kaur
Department of Microbiology, Khalsa College of Veterinary and Animal Sciences
Amritsar (143001), Punjab, India

Received on 16th August, 2019 (Accepted on 20th December, 2020).

ABSTRACT
Aspergillus fumigatus, an opportunistic fungal pathogen, is responsible for skin infection in both humans
and animals. The incidence of A. fumigatus dermatitis in canine as a primary pathogen is rarely reported. In
this study, A. fumigatus and A. flavus were isolated from clinical samples of skin scrapings and infected hair
from canines suffering from recurrent skin conditions. Sabouraud’s dextrose agar (SDA) supplemented with
chloramphenicol yielded typical velvety bluish-green colony of A. fumigatus and yellow-green cottony colony
with sugary texture of A. flavus. The susceptibility of these isolates against anti-fungal drugs (ketoconazole
and itraconazole) was evaluated by disk diffusion method. A. fumigatus was found resistant to ketoconazole
but susceptible to itraconazole, whereas, A. flavus is susceptible to both ketoconazole and itraconazole.
Antifungal susceptibility test for all fungal isolates should be routinely performed to prevent resistance against
antifungal drugs used in veterinary dermatitis cases.
Key words: Antifungal sensitivity test, Aspergillus, Recurrent skin disease, SDA

INTRODUCTION resistance to fluconazole and increased resistance

The genus Aspergillus has been classified into to the azole antifungal drugs (Van der Linden et al.,
8 distinct subgenera viz Aspergillus, Fumigati, 2011; Leonardelli et al., 2016; Wiederhold, 2017).
Circumdati, Terrei, Nidulantes, Ornati, Warcupi, and The widespread and increased use of azoles in the
Candidi (Peterson et al., 2008). More than 200 treatment of aspergillosis and their growing
known species exist in the genus, but only a small resistance to azoles group of drugs reiterate the
percentage are associated with infections which importance of antifungal susceptibility studies to
includes pathogens such as, Aspergillus fumigatus, understand the resistance profile and improve
A. flavus and A. niger (Greub&Bille, 1998). A. treatment.
terreus and A. versicolor are occasionally isolated A.fumigatus and A.flavus were both isolated
from clinical specimens. The infections ranges from from clinical samples of two dogs with chronic
localized skin/nail/ocular infection to pulmonary recurrent skin problem which could contribute to
disorder and invasive systemic infection collectively contamination of their environment with pathogenic
referred to as “aspergillosis,” (Zhang et al., 2012; fungal spores, causing great public health concern.
Veraldi et al., 2010; Ozer, et al., 2009; Peeters & The aim of this paper is to evaluate both these
Clercx, 2007; Sharp & Matthews, 2006). Three isolates and their antifungal susceptibility by disk
major forms of aspergillosis such as nasal, diffusion method against commonly used antifungal
bronchopulmonary, and disseminated infections agents in veterinary practice.
occur in dogs caused by the primary pathogen A.
fumigatus, followed closely by A. flavus and A. niger
(Seyedmousavi et al., 2015). However, incidence MATERIALS AND METHODS
of A. fumigatus cutaneous dermatitis in canine Sample collection:
species as a primary pathogen is rarely reported. The Hair and skin scraping were collected
treatment of choice for these disease conditions is aseptically from two canine patients (a spayed
based on the use of antifungal drugs such as female retriever-mixed and an intact rescued female
voriconazole, itraconazole, posaconazole, dog) with recurrent skin infection (Figs. 1 & 2).
ketoconazole, thiabendazole and more recently, Direct impression smears from the skin of both
isavuconazole (Denardi, 2018). Nevertheless, A. patients were also collected for preliminary staining
fumigatus has been reported to develop intrinsic with Lactophenol cotton blue (LPCB) stain and 10%

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 Aspergillus in dogs

spores. The conidia were mixed in sterile saline

solution and 0.05% Tween 20. Inoculum size was
adjusted to a volume of 2 x 106 CFU/ml using a
haemocytometer. Antifungal susceptibility against
itraconazole and ketoconazole was determined using
disk diffusion method on Mueller-Hinton Agar
(MHA) containing 2% glucose and 0.5 µg/ml of
methylene blue dye according to the guidelines of
Clinical and Laboratory Standards Institute (CLSI)-
M51-A (CLSI-M51-A).

RESULTS AND DISCUSSION

The initial staining of hair, skin scrapings and
impression smears from the two patients indicated
fungal spores of Aspergillus sp. Cutaneous
Fig. 1: Photograph of an intact female rescued mongrel aspergillosis is mainly caused by ubiquitous soil and
weighing 27 kgs, with skin lesion on the ventral abdomen,
water-dwelling saprophytes of the genus in
caudal thighs, tail-head, neck extending to lateral chest and
abdomen, face and over the dorsum of the body. (Patient immunocompromised patients. Although, skin
no. 1) infection caused by A. fumigatus, as a primary
pathogen, has not been reported in dogs, A. flavus
and A. terreus are commonly isolated. It has also
been reported that outbreaks of Aspergillosis
involving skin, oral mucosa or subcutaneous tissues
are more often associated with A. flavusthan other
species (Heinemann et al., 2004; Hedayati et al.,
2007). In this study, two dogs showed similar
cutaneous symptoms with formation of papules and
nodules on the affected area of the body. Hedayati
et al. (2007) also reported the presence of
hemorrhagic bullae, ulcerations with central necrosis
Fig. 2: Photograph of a spayed female retriever-mixed
weighing 28kgs with generalized papulonodular skin lesions with or without eschar formation, pustules or
on forelimbs and hindlimbs.(Patient no, 2) subcutaneous abscesses in A. flavus in skin
infections.
KOH solution to confirm the presence of fungal Isolation of fungus on DTM yield no growth
spores. even after 5 days of incubation, hence kept for
further incubation at the required temperature and
Isolation of fungus: humidity. Inoculation of skin scrapings in SDA
Hair and skin scraping samples were plates supplemented with chloramphenicol yielded
inoculated on three separate plates of Sabouraud’s mixed colony of A. flavus and A. fumigatus (Fig.
Dextrose Agar (SDA) supplemented with 3a,3b& 4a). A. fumigatus is a fast grower; the colony
chloramphenicol (0.05mg/l) and Dermatophyte test size reached 3 cm within 5 days with a typical
medium (DTM) to rule out dermatophytes infection. velvety bluish-green colony (Fig.3c). A. flavus
The inoculated plates were incubated at 25°C and showed velvety yellow color colony with a sugary
37°C, respectively, for 5 days with sufficient texture, and reached 4 cm in size within 18 days of
humidity. Fungal colonies were identified based on incubation (Fig. 3d). Microscopic examination of
their colony characteristics and microscopic the morphology of conidia and conidiophores
morphology of conidia and conidiophores as per confirmed the typical the bluish-green color of A.
standard protocols (Markey et al., 2013). fumigatus conidial heads and the clearly distinct
yellowish-green conidial heads of A. flavus. The
Antifungal susceptibility test: bluish-green color conidia were arranged in chains
Five isolated colonies of A. fumigatus and A. basipetally from greenish phialids borne directly on
flavus from clinical samples were further broadly clavate vesicles (20 to 30 mm in diameter)
subcultured on SDA to obtain pure and adequate (Fig. 4b). A. flavus, on the other hand, showed

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Ralte et al.

Fig. 5: Antifungal sensitivity test (a) A.fumigatus with

resistance to ketoconazole. (b) A. flavus showed sensitivity
to both ketoconazole and itraconazol

testing and to investigate the impact of elevated

minimum inhibitory concentrations on antifungal
drug efficacy.
Table 1: Antifungal sensitivity test by disk diffusion
Fig. 3: Fungal colony consisting of A. fumigatus and A.
method (CLSI-M51-A)
flavus (a)mixed colonyisolated from skin scraping of patient
no. 1 (b)mixed colony isolated from skin scraping of patient Fungal Isolates Ketoconazole Itraconazole
no. 2. (c)purecolony of A. fumigatus isolated from hair (KT) (IT)
sample of patient no.1 (d) pure colony of A. flavus isolated A. fumigatus R S
from hair sample of patient no. 2
A. flavus S S
R= resistant; S= sensitivity

CONCLUSIONS
Isolation of Aspergillus species from
superficial clinical samples indicatedits
pathogenicity to animals with an immune
compromised system. There is also the possibility
Fig. 4: Lacto-phenol cotton blue stain of colonies, 400X of pet dogs in spreading fungal spores within their
(a) Mixed A. fumigatus and A. flavus isolated from skin
scrapings of patient no. 1 and 2. (b) A. fumigatus isolated
immediate environment. Furthermore, antifungal
from hair sample of patient no. 1 (c) A. flavus isolated from susceptibility test for all fungal isolates should be
hair sample of patient no. 2. routinely performed to evade an increase in fungal
resistance to the limited antifungal drugs used in
distinct yellowish-green radiating conidial heads veterinary practice. For specific therapeutic dosage,
arranged in chains from phialids which arised minimum inhibition concentration (MIC) of each
circumferentially from a globose vesicle (Fig. 4c). sensitive drug should be performed further.
(Markey et al., 2013).
Species differentiation is clinically important ACKNOWLEDGEMENT
for selection of appropriate antifungal drugs (Lass- We thank the Principal, Khalsa College of
Flöri, 2014). Antifungal sensitivity test of A. Veterinary and Animal Sciences, Amritsar for
fumigatus showed resistant to ketoconazole but providing the necessary facilities required for our
susceptible to itraconazole. (Fig. 5a). Whereas, A. work.
flavus was found susceptible to both ketoconazole
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