- Email: [email protected]
Fungal Dermatitis Caused by Aspergillus sydowii in a Thoroughbred Horse
Journal of Equine Veterinary Science 32 (2012) 835-839
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Case Report
Fungal Dermatitis Caused by Aspergillus sydowii in a Thoroughbred Horse Sang Kyu Lee DVM a, b, Hyun-Gu Kang DVM, PhD b, Ki-Jeong Na DVM, PhD b, Jae-Ik Han DVM, PhD b, c a
Veterinary Center, Korea Racing Authority, Gwacheon, South Korea College of Veterinary Medicine, Chungbuk National University, Cheongju, South Korea c Research Institute of Veterinary Medicine, Chungbuk National University, Cheongju, South Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 January 2012 Received in revised form 6 March 2012 Accepted 21 March 2012 Available online 26 April 2012
A 5-year-old male Thoroughbred horse was referred with alopecia, along with severe incrustation, scaling, and pruritus on the left flank and back, which had been in contact with a saddle for 1 year. Treatment with topical antibiotics by the owner was not effective. A fungal culture using the hair roots and exudates under the scabs showed blue-green colonies surrounded by a white band. Microscopic examination and molecular analyses of the internal transcribed spacer region, partial b-tubulin, and calmodulin genes confirmed this fungus as Aspergillus sydowii. Topical treatment with econazole ointment for 2 months was effective. This is the first report of a natural A sydowii infection in a horse. Ó 2012 Elsevier Inc. All rights reserved.
Keywords: Aspergillus sydowii Dermatitis Horse Molecular typing
1. Introduction Aspergillus species are ubiquitous molds found in soil, water, and decaying materials, and appear as white to green, fluffy, and sporulating molds [1]. Generally, they are not pathogenic; however, Aspergillus species infection continues to cause serious diseases in immunocompromised humans and animals [2]. Most Aspergillus infections are caused by Aspergillus fumigatus and Aspergillus flavus [3]. In horses, infection caused by Aspergillus species is most commonly seen as guttural pouch mycosis [4,5]. Because of the hemorrhaging caused by gradual erosion of the arterial wall by fungal growth, guttural pouch mycosis is considered a life-threatening opportunistic infection in adult immunocompetent horses. Infection caused by Aspergillus species is also associated with nasal cavity, lung, mediastinum, placenta, eye, and uterus in horses [6-11]. Corresponding author at: Jae-Ik Han, DVM, PhD, Research Institute of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, South Korea. E-mail address: [email protected] (J.-I. Han). 0737-0806/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2012.03.013
Cutaneous aspergillosis may be classified as either primary or secondary, depending on the underlying pathogenesis [12]. Cases in which the initial infection begins at the site of skin injury are classified as primary cutaneous aspergillosis, whereas secondary cutaneous aspergillosis results from the extension of the infection to the skin either from other infected sites or because of hematogenous spreading. Unlike dermatophytes, which release keratinolytic proteases, Aspergillus species cannot use epidermal and hair follicle keratin; therefore, Aspergillus species require a disruption of the keratin structure to cause infection [13,14]. After disruption by trauma or maceration, conidia of the Aspergillus species germinate into hyphae and invade the deep layers of the skin. Occasionally, the hyphae invade blood vessels; therefore, primary cutaneous aspergillosis can result in disseminated aspergillosis, especially in immunocompromised hosts. This report describes a case of fungal dermatitis caused by A sydowii diagnosed by microscopic examination, fungal culture, and molecular analysis in a Thoroughbred horse. To our knowledge, this is the first case report of natural A sydowii infection in the horse.
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S.K. Lee et al. / Journal of Equine Veterinary Science 32 (2012) 835-839
Fig. 1. The horse exhibited well-circumscribed, complete alopecia, with severe incrustation and pruritus. Many hair were separated from the hair follicles and only attached to the scabs on the lesion and were, therefore, easily epilated.
2. Case Description 2.1. Clinical History A 5-year-old male Thoroughbred horse developed a refractory alopecia with incrustation on its left flank and back. Clinical signs were noted for >1 year prior to referral. The horse had been treated with a 2.5% topical chloramphenicol spray (Pinkskin; Bayer Animal Health Co., Suwon, South Korea) and Fucidin ointment (Dong Wha Pharm Co., Seoul, South Korea) for 10 months by the owner, but its condition continued to deteriorate. The horse was purchased from a racetrack for riding purposes 1 year earlier and was housed alone on straw bedding in a horse stable. It was fed commercial horse feed and alfalfa three times a day. At the first presentation, the horse exhibited wellcircumscribed, widely located alopecia on the left flank, accompanied by severe incrustation, scaling, and pruritus (Fig. 1). The lesions marginated the areas that were in contact with the saddle. The activity, appetite, defecation, and urination of the horse were unaffected. Samples were obtained by scraping scabs and plucking hair attached to the margins of the alopecic lesion and then analyzed by microscopic examination and fungal culture. The collected debris was smeared on a clean glass slide and stained with Wright-Giemsa stain, and results indicated that the smear contained many keratinized epithelial cells and septate hyphae. As such, daily topical application of econazole ointment (Ecocilon-G cream; Sky New Pharm, Siheung,
Fig. 2. Macro- and microscopic appearance of A sydowii cultured on Sabouraud dextrose agar. (A) Morphology of the cultured colony after 3 days at 30 C. (B) Conidial heads of the cultured colony showing typical features of Aspergillus species using Diff-Quik stain (400).
South Korea) was initiated as an empirical antifungal therapy on day 1 of presentation. By day 7, the incrustation and scaling had not improved, although the pruritus had disappeared. After 30 days of therapy, the incrustation and scaling had disappeared and hair had begun to regrow on the lesion. After 60 days of therapy, the lesion had disappeared and hair regrowth was complete. 2.2. Molecular Identification of the Fungal Isolate To confirm the fungal infection, hair roots and exudates collected under the scabs were cultured at 30 C for 10 days on Sabouraud dextrose agar. The cultured colonies were blue-green and surrounded by a white band. They were placed on a clean glass slide and stained using Diff-Quik stain (Sysmex, Hyogo, Japan). On microscopic analysis of the slide, conidial heads, similar to those seen in Aspergillus spp., were observed in the isolate (Fig. 2).
S.K. Lee et al. / Journal of Equine Veterinary Science 32 (2012) 835-839
837
Fig. 3. Phylogenetic tree based on the b-tubulin (A) and calmodulin (B) gene sequences of type strains in GenBank databases. Diamonds indicate sequences from the fungus isolated in this case. The first series of letters/numbers represents GenBank database numbers, and the second series of letters/numbers represents the numbers of the type strains. Sequence alignments were performed using CLUSTAL-X v. 1.8 (UCD Conway Institute, Dublin, Ireland), and phylogenetic analyses were performed using MEGA4 v. 4.0.2 (Tempe, AZ, USA).
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S.K. Lee et al. / Journal of Equine Veterinary Science 32 (2012) 835-839
The isolate was then identified by molecular typing. The genomic DNA encoding the ribosomal internal transcribed spacer (ITS) region, consisting of ITS1, 5.8S, and ITS2, was amplified using primers ITS1 and ITS4, as described previously [15]. Following previously described protocols, partial b-tubulin and calmodulin genes were also partially amplified using primers Bt2a and Bt2b [16], and primers Cmd5 and Cmd6 [17], respectively. All amplicons were sequenced using an ABI prism Bigdye Terminator Cycle Sequencing Ready reaction kit V.3.1 (PE Applied Biosystems, Foster City, CA). When compared with the sequences in GenBank database, the ITS sequence of the isolate was 100% similar to the A sydowii sequence deposited by the University of Puerto Rico, Puerto Rico (GenBank accession number EU645721), the b-tubulin sequence was found to be 100% similar to the A sydowii sequence deposited by Agriculture and Agri-Food Canada (accession number EU907905), and the calmodulin sequence was found to be 99% similar to the A sydowii sequence deposited by the National Center for Agricultural Utilization Research, United States (accession number EF652385). Therefore, we identified the species isolated from the horse as A sydowii. Phylogenetic analyses of the nucleotide sequences of the b-tubulin and calmodulin genes also confirmed the results of our molecular identification (Fig. 3).
of dermatophytosis, and the fungal colonies during the early stages of culture were white, we initially suspected this to be a case of dermatophytosis. However, the color of the cultured colonies became green with white margins during the later stages of culture, and microscopic examination revealed that the conidial heads of the isolate resembled those of Aspergillus species. Thus, we performed molecular typing of the isolate to ensure accurate identification. Traditionally, the identification of Aspergillus species has been performed based on the macro- and microscopic examinations. Those procedures are reliable; however, they are not appropriate for clinical diagnostic laboratories because they are time-consuming and require specialized knowledge [25,26]. Instead, the availability of the molecular typing, including ITS, b-tubulin, calmodulin, and actin genes, has been proved for species identification and phylogenetic analysis [27,28]. Thus, we performed the molecular typing using ITS, b-tubulin, and calmodulin followed by phylogenetic analyses, and confirmed that A sydowii caused the fungal dermatitis in our case. Although fungal infection is often suspected in clinical cases, the use of molecular test in early step will allow us to predict the prognosis and to decide therapeutic choices.
3. Discussion
The ITS region, calmodulin, and b-tubulin nucleotide sequences of the isolate were deposited in Genbank under accession numbers HM061186, HM061187, and HM061188, respectively. Living cultures of the case strain were deposited in the Korean Collection for Type Culture of the Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea (no. KCTC26432).
The fungus A sydowii is a mesophilic soil-borne saprobe, which is distributed worldwide as a food contaminant [18]. Like other Aspergillus species, A sydowii can be an opportunistic pathogen in humans, depending on the host’s immune status and the extent and duration of exposure. Historically, A sydowii has been associated with onychomycosis, root canal infection, and peritonitis secondary to peritoneal dialysis in humans [19-21]. Although Aspergillus species, including A sydowii, are not normal inhabitants of marine environments, A sydowii can become a pathogen in sea fan corals in warm water temperatures, which promote growth of the fungus while reducing the efficacy of host resistance [18]. It is noteworthy that when Aspergillus species are isolated from a lesion, it can be difficult to determine whether the fungus is the actual pathogen or just a contaminant from the environment. In this case, we diagnosed the fungal dermatitis caused by A sydowii from the following observations: (i) topical antibiotics were not effective; (ii) microscopic examination of the scraped debris revealed the presence of septate hyphae; (iii) cultures of the exudates under the scabs and the hair roots showed the growth of A sydowii but no other fungi; (iv) a topical antifungal was effective; and (v) molecular analyses of the isolate’s ITS region, b-tubulin, and calmodulin genes showed complete identity with the nucleotide sequences of A sydowii, and no DNA of any other fungus was detected. In animals, fungal dermatitis resulting in alopecia, hyperkeratosis, and scaling is usually caused by dermatophytes [22,23], with Trichophyton equinum being the most common dermatophyte in horses [24]. Dermatophytes cultured on Sabouraud dextrose agar usually have white to yellowish white colonies with variable appearance [23]. Because the clinical lesion described here was similar to that
Acknowledgments
References [1] Collier L, Balows A, Sussman M, Ajello L, Hay RJ. Topley and Wilson’s Microbiology and Microbial Infection. 9th ed. New York, NY: Oxford University Press; 1998. [2] Venner M, Reinhold B, Beyerbach M, Feige K. Efficacy of azithromycin in preventing pulmonary abscesses in foals. Vet J 2009;179:301-3. [3] Pasqualotto AC. Difference in pathogenicity and clinical syndromes due to Aspergillus fumigatus and Aspergillus flavus. Med Mycol 2009; 47:S261-70. [4] Ludwig A, Gatineau S, Reynaud MC, Cadoré JL, Bourdoiseau G. Fungal isolation and identification in 21 cases of guttural pouch mycosis in horses (1998-2002). Vet J 2005;169:457-61. [5] Tell LA. Aspergillosis in mammals and birds: impact on veterinary medicine. Med Mycol 2005;43:S71-3. [6] Pugh DG, Bowen JM, Kloppe LH, Simpson RB. Fungal endometritis in mares. Comp Cont Educ Pract Vet 1986;8:S173-81. [7] Samuelson DA, Andresen TL, Gwin RM. Conjunctival fungal flora in horses, cattle, dogs, and cats. J Am Vet Med Assoc 1984;184:1240-2. [8] Blue J, Perdrizet J, Brown E. Pulmonary aspergillosis in a horse with myelomonocytic leukemia. J Am Vet Med Assoc 1987;190:1562-4. [9] Greet TRC. Nasal aspergillosis in three horses. Vet Rec 1981;109:487-9. [10] Mahaffey LW, Adam NM. Abortions associated with mycotic lesions of the placenta in mares. J Am Vet Med Assoc 1964;144:24-32. [11] Rush-Moore B, Reed SM, Kowalski JJ, Bertone JJ. Aspergillosis granuloma in the mediastinum of a immunocompromised horse. Cornell Vet 1993;83:97-104. [12] Van Burik JH, Colven R, Spach DH. Cutaneous aspergillosis. J Clin Microbiol 1998;36:3115-21. [13] Vermout S, Tabart J, Baldo A, Mathy A, Losson B, Mignon B. Pathogenesis of dermatophytosis. Mycopathologia 2008;166:267-75. [14] Walsh TJ. Editorial response: primary cutaneous aspergillosis-an emerging infection among immunocompromised patients. Clin Infect Dis 1998;27:453-7. [15] Makimura K, Mochizuki T, Hasegawa A, Uchida K, Saito H, Tamaguchi H. Phylogenetic classification of Trichophyton mentagrophytes complex
S.K. Lee et al. / Journal of Equine Veterinary Science 32 (2012) 835-839
[16]
[17]
[18]
[19]
[20] [21]
strains based on DNA sequences of nuclear ribosomal internal transcribed spacer 1 regions. J Clin Microbiol 1998;36:2629-33. Glass NL, Donaldson GC. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol 1995;61:1323-30. Hong SB, Go SJ, Shin HD, Frisvad JC, Samson RA. Polyphasic taxonomy of Aspergillus fumigatus and related species. Mycologia 2005;97:1342-55. Alker AP, Smith GW, Kim K. Characterization of Aspergillus sydowii (Thom et Church), a fungal pathogen of Caribbean sea fan corals. Hydrobiologia 2001;460:105-11. Takahata Y, Hiruma M, Sugita T, Muto M. A case of onychomycosis due to Aspergillus sydowii diagnosed using DNA sequence analysis. Mycoses 2007;51:170-3. Gomes C, Fidel S, Fidel R, de Moura Sarquis MI. Isolation and taxonomy of filamentous fungi in endodontic infections. J Endod 2010;36:626-9. Chiu YL, Liaw SJ, Wu VC, Hsueh PR. Peritonitis caused by Aspergillus sydowii in a patient undergoing continuous ambulatory peritoneal dialysis. J Infect 2005;51:e159-61.
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[22] Scott DW, Miller WH, eds. Fungal skin diseases. In: Equine dermatology. 1st ed. Philadelphia, PA: Saunders; 2001: p. 261-320. [23] Scott DW, Miller WH, Griffin CE, eds. Superficial mycoses: fungal skin disease. In: Muller and Kirk’s small animal dermatology. Philadelphia, PA: Saunders; 2001: p. 338-61. [24] Mahmoud AL. Dermatophytes and other keratinophilic fungi causing ringworm of horses. Folia Microbiol 1995;40:293-6. [25] Samson RA, Hong SB, Frisvad JC. Old and new concepts of species differentiation in Aspergillus. Med Mycol 2006;44:133-48. [26] Guarro J, Gene J, Stchigel AM. Developments in fungal taxonomy. Clin Microbiol 1999;12:454-500. [27] Hinrikson HP, Hurst SF, Lott TJ, Warnock DW, Morrison CJ. Assessment of ribosomal large-subunit D1-D2, internal transcribed spacer 1, and internal transcribed spacer 2 regions as targets for molecular identification of medically important Aspergillus species. J Clin Microbiol 2005;43:2092-103. [28] Samson RA, Hong SB, Frisvad JC, Varga J. Polyphasic taxonomy of Aspergillus section Fumigati and its teleomorph Neosartorya. Stud Mycol 2007;59:147-203.
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Case Report
Fungal Dermatitis Caused by Aspergillus sydowii in a Thoroughbred Horse Sang Kyu Lee DVM a, b, Hyun-Gu Kang DVM, PhD b, Ki-Jeong Na DVM, PhD b, Jae-Ik Han DVM, PhD b, c a
Veterinary Center, Korea Racing Authority, Gwacheon, South Korea College of Veterinary Medicine, Chungbuk National University, Cheongju, South Korea c Research Institute of Veterinary Medicine, Chungbuk National University, Cheongju, South Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 January 2012 Received in revised form 6 March 2012 Accepted 21 March 2012 Available online 26 April 2012
A 5-year-old male Thoroughbred horse was referred with alopecia, along with severe incrustation, scaling, and pruritus on the left flank and back, which had been in contact with a saddle for 1 year. Treatment with topical antibiotics by the owner was not effective. A fungal culture using the hair roots and exudates under the scabs showed blue-green colonies surrounded by a white band. Microscopic examination and molecular analyses of the internal transcribed spacer region, partial b-tubulin, and calmodulin genes confirmed this fungus as Aspergillus sydowii. Topical treatment with econazole ointment for 2 months was effective. This is the first report of a natural A sydowii infection in a horse. Ó 2012 Elsevier Inc. All rights reserved.
Keywords: Aspergillus sydowii Dermatitis Horse Molecular typing
1. Introduction Aspergillus species are ubiquitous molds found in soil, water, and decaying materials, and appear as white to green, fluffy, and sporulating molds [1]. Generally, they are not pathogenic; however, Aspergillus species infection continues to cause serious diseases in immunocompromised humans and animals [2]. Most Aspergillus infections are caused by Aspergillus fumigatus and Aspergillus flavus [3]. In horses, infection caused by Aspergillus species is most commonly seen as guttural pouch mycosis [4,5]. Because of the hemorrhaging caused by gradual erosion of the arterial wall by fungal growth, guttural pouch mycosis is considered a life-threatening opportunistic infection in adult immunocompetent horses. Infection caused by Aspergillus species is also associated with nasal cavity, lung, mediastinum, placenta, eye, and uterus in horses [6-11]. Corresponding author at: Jae-Ik Han, DVM, PhD, Research Institute of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, South Korea. E-mail address: [email protected] (J.-I. Han). 0737-0806/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2012.03.013
Cutaneous aspergillosis may be classified as either primary or secondary, depending on the underlying pathogenesis [12]. Cases in which the initial infection begins at the site of skin injury are classified as primary cutaneous aspergillosis, whereas secondary cutaneous aspergillosis results from the extension of the infection to the skin either from other infected sites or because of hematogenous spreading. Unlike dermatophytes, which release keratinolytic proteases, Aspergillus species cannot use epidermal and hair follicle keratin; therefore, Aspergillus species require a disruption of the keratin structure to cause infection [13,14]. After disruption by trauma or maceration, conidia of the Aspergillus species germinate into hyphae and invade the deep layers of the skin. Occasionally, the hyphae invade blood vessels; therefore, primary cutaneous aspergillosis can result in disseminated aspergillosis, especially in immunocompromised hosts. This report describes a case of fungal dermatitis caused by A sydowii diagnosed by microscopic examination, fungal culture, and molecular analysis in a Thoroughbred horse. To our knowledge, this is the first case report of natural A sydowii infection in the horse.
836
S.K. Lee et al. / Journal of Equine Veterinary Science 32 (2012) 835-839
Fig. 1. The horse exhibited well-circumscribed, complete alopecia, with severe incrustation and pruritus. Many hair were separated from the hair follicles and only attached to the scabs on the lesion and were, therefore, easily epilated.
2. Case Description 2.1. Clinical History A 5-year-old male Thoroughbred horse developed a refractory alopecia with incrustation on its left flank and back. Clinical signs were noted for >1 year prior to referral. The horse had been treated with a 2.5% topical chloramphenicol spray (Pinkskin; Bayer Animal Health Co., Suwon, South Korea) and Fucidin ointment (Dong Wha Pharm Co., Seoul, South Korea) for 10 months by the owner, but its condition continued to deteriorate. The horse was purchased from a racetrack for riding purposes 1 year earlier and was housed alone on straw bedding in a horse stable. It was fed commercial horse feed and alfalfa three times a day. At the first presentation, the horse exhibited wellcircumscribed, widely located alopecia on the left flank, accompanied by severe incrustation, scaling, and pruritus (Fig. 1). The lesions marginated the areas that were in contact with the saddle. The activity, appetite, defecation, and urination of the horse were unaffected. Samples were obtained by scraping scabs and plucking hair attached to the margins of the alopecic lesion and then analyzed by microscopic examination and fungal culture. The collected debris was smeared on a clean glass slide and stained with Wright-Giemsa stain, and results indicated that the smear contained many keratinized epithelial cells and septate hyphae. As such, daily topical application of econazole ointment (Ecocilon-G cream; Sky New Pharm, Siheung,
Fig. 2. Macro- and microscopic appearance of A sydowii cultured on Sabouraud dextrose agar. (A) Morphology of the cultured colony after 3 days at 30 C. (B) Conidial heads of the cultured colony showing typical features of Aspergillus species using Diff-Quik stain (400).
South Korea) was initiated as an empirical antifungal therapy on day 1 of presentation. By day 7, the incrustation and scaling had not improved, although the pruritus had disappeared. After 30 days of therapy, the incrustation and scaling had disappeared and hair had begun to regrow on the lesion. After 60 days of therapy, the lesion had disappeared and hair regrowth was complete. 2.2. Molecular Identification of the Fungal Isolate To confirm the fungal infection, hair roots and exudates collected under the scabs were cultured at 30 C for 10 days on Sabouraud dextrose agar. The cultured colonies were blue-green and surrounded by a white band. They were placed on a clean glass slide and stained using Diff-Quik stain (Sysmex, Hyogo, Japan). On microscopic analysis of the slide, conidial heads, similar to those seen in Aspergillus spp., were observed in the isolate (Fig. 2).
S.K. Lee et al. / Journal of Equine Veterinary Science 32 (2012) 835-839
837
Fig. 3. Phylogenetic tree based on the b-tubulin (A) and calmodulin (B) gene sequences of type strains in GenBank databases. Diamonds indicate sequences from the fungus isolated in this case. The first series of letters/numbers represents GenBank database numbers, and the second series of letters/numbers represents the numbers of the type strains. Sequence alignments were performed using CLUSTAL-X v. 1.8 (UCD Conway Institute, Dublin, Ireland), and phylogenetic analyses were performed using MEGA4 v. 4.0.2 (Tempe, AZ, USA).
838
S.K. Lee et al. / Journal of Equine Veterinary Science 32 (2012) 835-839
The isolate was then identified by molecular typing. The genomic DNA encoding the ribosomal internal transcribed spacer (ITS) region, consisting of ITS1, 5.8S, and ITS2, was amplified using primers ITS1 and ITS4, as described previously [15]. Following previously described protocols, partial b-tubulin and calmodulin genes were also partially amplified using primers Bt2a and Bt2b [16], and primers Cmd5 and Cmd6 [17], respectively. All amplicons were sequenced using an ABI prism Bigdye Terminator Cycle Sequencing Ready reaction kit V.3.1 (PE Applied Biosystems, Foster City, CA). When compared with the sequences in GenBank database, the ITS sequence of the isolate was 100% similar to the A sydowii sequence deposited by the University of Puerto Rico, Puerto Rico (GenBank accession number EU645721), the b-tubulin sequence was found to be 100% similar to the A sydowii sequence deposited by Agriculture and Agri-Food Canada (accession number EU907905), and the calmodulin sequence was found to be 99% similar to the A sydowii sequence deposited by the National Center for Agricultural Utilization Research, United States (accession number EF652385). Therefore, we identified the species isolated from the horse as A sydowii. Phylogenetic analyses of the nucleotide sequences of the b-tubulin and calmodulin genes also confirmed the results of our molecular identification (Fig. 3).
of dermatophytosis, and the fungal colonies during the early stages of culture were white, we initially suspected this to be a case of dermatophytosis. However, the color of the cultured colonies became green with white margins during the later stages of culture, and microscopic examination revealed that the conidial heads of the isolate resembled those of Aspergillus species. Thus, we performed molecular typing of the isolate to ensure accurate identification. Traditionally, the identification of Aspergillus species has been performed based on the macro- and microscopic examinations. Those procedures are reliable; however, they are not appropriate for clinical diagnostic laboratories because they are time-consuming and require specialized knowledge [25,26]. Instead, the availability of the molecular typing, including ITS, b-tubulin, calmodulin, and actin genes, has been proved for species identification and phylogenetic analysis [27,28]. Thus, we performed the molecular typing using ITS, b-tubulin, and calmodulin followed by phylogenetic analyses, and confirmed that A sydowii caused the fungal dermatitis in our case. Although fungal infection is often suspected in clinical cases, the use of molecular test in early step will allow us to predict the prognosis and to decide therapeutic choices.
3. Discussion
The ITS region, calmodulin, and b-tubulin nucleotide sequences of the isolate were deposited in Genbank under accession numbers HM061186, HM061187, and HM061188, respectively. Living cultures of the case strain were deposited in the Korean Collection for Type Culture of the Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea (no. KCTC26432).
The fungus A sydowii is a mesophilic soil-borne saprobe, which is distributed worldwide as a food contaminant [18]. Like other Aspergillus species, A sydowii can be an opportunistic pathogen in humans, depending on the host’s immune status and the extent and duration of exposure. Historically, A sydowii has been associated with onychomycosis, root canal infection, and peritonitis secondary to peritoneal dialysis in humans [19-21]. Although Aspergillus species, including A sydowii, are not normal inhabitants of marine environments, A sydowii can become a pathogen in sea fan corals in warm water temperatures, which promote growth of the fungus while reducing the efficacy of host resistance [18]. It is noteworthy that when Aspergillus species are isolated from a lesion, it can be difficult to determine whether the fungus is the actual pathogen or just a contaminant from the environment. In this case, we diagnosed the fungal dermatitis caused by A sydowii from the following observations: (i) topical antibiotics were not effective; (ii) microscopic examination of the scraped debris revealed the presence of septate hyphae; (iii) cultures of the exudates under the scabs and the hair roots showed the growth of A sydowii but no other fungi; (iv) a topical antifungal was effective; and (v) molecular analyses of the isolate’s ITS region, b-tubulin, and calmodulin genes showed complete identity with the nucleotide sequences of A sydowii, and no DNA of any other fungus was detected. In animals, fungal dermatitis resulting in alopecia, hyperkeratosis, and scaling is usually caused by dermatophytes [22,23], with Trichophyton equinum being the most common dermatophyte in horses [24]. Dermatophytes cultured on Sabouraud dextrose agar usually have white to yellowish white colonies with variable appearance [23]. Because the clinical lesion described here was similar to that
Acknowledgments
References [1] Collier L, Balows A, Sussman M, Ajello L, Hay RJ. Topley and Wilson’s Microbiology and Microbial Infection. 9th ed. New York, NY: Oxford University Press; 1998. [2] Venner M, Reinhold B, Beyerbach M, Feige K. Efficacy of azithromycin in preventing pulmonary abscesses in foals. Vet J 2009;179:301-3. [3] Pasqualotto AC. Difference in pathogenicity and clinical syndromes due to Aspergillus fumigatus and Aspergillus flavus. Med Mycol 2009; 47:S261-70. [4] Ludwig A, Gatineau S, Reynaud MC, Cadoré JL, Bourdoiseau G. Fungal isolation and identification in 21 cases of guttural pouch mycosis in horses (1998-2002). Vet J 2005;169:457-61. [5] Tell LA. Aspergillosis in mammals and birds: impact on veterinary medicine. Med Mycol 2005;43:S71-3. [6] Pugh DG, Bowen JM, Kloppe LH, Simpson RB. Fungal endometritis in mares. Comp Cont Educ Pract Vet 1986;8:S173-81. [7] Samuelson DA, Andresen TL, Gwin RM. Conjunctival fungal flora in horses, cattle, dogs, and cats. J Am Vet Med Assoc 1984;184:1240-2. [8] Blue J, Perdrizet J, Brown E. Pulmonary aspergillosis in a horse with myelomonocytic leukemia. J Am Vet Med Assoc 1987;190:1562-4. [9] Greet TRC. Nasal aspergillosis in three horses. Vet Rec 1981;109:487-9. [10] Mahaffey LW, Adam NM. Abortions associated with mycotic lesions of the placenta in mares. J Am Vet Med Assoc 1964;144:24-32. [11] Rush-Moore B, Reed SM, Kowalski JJ, Bertone JJ. Aspergillosis granuloma in the mediastinum of a immunocompromised horse. Cornell Vet 1993;83:97-104. [12] Van Burik JH, Colven R, Spach DH. Cutaneous aspergillosis. J Clin Microbiol 1998;36:3115-21. [13] Vermout S, Tabart J, Baldo A, Mathy A, Losson B, Mignon B. Pathogenesis of dermatophytosis. Mycopathologia 2008;166:267-75. [14] Walsh TJ. Editorial response: primary cutaneous aspergillosis-an emerging infection among immunocompromised patients. Clin Infect Dis 1998;27:453-7. [15] Makimura K, Mochizuki T, Hasegawa A, Uchida K, Saito H, Tamaguchi H. Phylogenetic classification of Trichophyton mentagrophytes complex
S.K. Lee et al. / Journal of Equine Veterinary Science 32 (2012) 835-839
[16]
[17]
[18]
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