Clinical manifestation of an amelanotic Sporothrix schenckii complex isolate in a cat in Germany

Correspondence Sandra Scheufen Sandra.Scheufen@vetmed.unigiessen.de Institute of Hygiene and Infectious Diseases of Animals, Justus-Liebig-University Giessen, Giessen, Germany Clinic for Small Animals, Department of Veterinary Clinical Sciences, Justus-Liebig-University Giessen, Giessen, Germany Institute for Ecology, Evolution and Diversity, Department of Mycology, J W Goethe-University, Frankfurt am Main, Germany Institute for Veterinary Pathology, Justus-Liebig-University Giessen, Giessen, Germany


Introduction
Sporothrix schenckii is a dimorphic fungus well known as the causative agent of sporotrichosis in humans and animals.Besides its presence as a geophilic fungus, sporotrichosis has been reported in many different animal species with cats being suspected as the most prominent vector in transmission of the fungal infection to humans (Schubach et al., 2005).One of the crucial pathogenic mechanisms of dimorphic fungi is their ability to change from a mycelial form at temperatures lower than 30 uC into a thermotolerant phase with yeast-like structures, which are capable of causing fixed cutaneous or occasionally systemic disease after infecting a host via wounds or scratches (Rodrigues et al., 2013a;Welsh, 2003).Besides the dimorphism, melanization of conidia and the route of infection are regarded as important aspects in terms of the clinical relevance of a strain, as a correlation with the degree of virulence has been observed (Arrillaga-Moncrieff et al., 2009;Barros et al., 2011;Brito et al., 2007;Madrid et al., 2010b;Nobre et al., 2005).
Based on DNA analysis of the internal transcribed spacer (ITS) region and the calmodulin gene, the S. schenckii complex comprises a variety of strains usually divided into clades, which refer to their geographical distribution and their assumed medical importance (Rodrigues et al., 2014;Zhou et al., 2013).The following strains are usually regarded as clinically important members of the complex: Sporothrix brasiliensis, Sporothrix globosa, Sporothrix luriei, Sporothrix mexicana and Sporothrix schenckii sensu stricto (Marimon et al., 2006(Marimon et al., , 2007;;Zhou et al., 2013).Except for S. mexicana, the four other species of the complex have been isolated primarily from clinical cases and therefore differ from environmental strains with a low potential to cause infection in mammals, such as Sporothrix pallida and Sporothrix stylites (Rodrigues et al., 2013b;Zhou et al., 2013).Furthermore, a correlation between species with higher virulence and geographical distribution has been supposed.Clinically relevant species of S. schenckii, for example, have a main focus in Brazil, whereas in Germany predominantly environmental species are mentioned (Zhou et al., 2013).Regarding feline sporotrichosis, only a few cases have been reported in Germany (Weingart et al., 2010).
The present case report of feline sporotrichosis caused by a member of the Sporothrix schenckii complex demonstrates the importance of considering amelanotic members of the complex in cutaneous lesions of cats, even in regions with a low prevalence of sporotrichosis.

Case report
Between March and August 2013, a 10-year-old rescued Persian-mixed breed cat was repeatedly presented to a university clinic for small animals due to a history of nasal discharge with involvement of initially the right and later the left eye after a fight with another cat in November 2012.Treatments with marbofloxacin and dexamethasone had shown no improvement.Instead, the nasal discharge worsened and became infiltrated with blood.According to the owner of the cat, there has been no stay abroad.

Clinical examination
Clinical examination of the cat revealed no severe abnormalities apart from repetitive sneezing and a nasal stridor as well as serous to haemorrhagic discharge from both nostrils.Along the bridge of the nose, a central dent was ascertainable.The right eye showed symptoms of mild conjunctivitis, viscous discharge and a dense mass measuring 0.5|0.5 cm on the nasal right orbita.Specific ophthalmological examination revealed a mild conjunctivitis of both eyes with epiphora particularly of the right eye and an entropium of the lower eyelids.Further investigation of the area around the prominent mass by computed tomography scan showed a severe soft-tissue swelling with infiltration of the nasal cavity and destruction of the right os nasale, parts of the ossa maxillaria and the septum nasi.
For laboratory examination including cytology, histopathology and microbiology, blood and biopsy samples from the right side of the nasal bridge were taken and a FeLV test was performed (SNAP Combo Plus FeLV Ag/FIV Ab test; IDEXX Laboratories).

Cytology and histopathology
For cytological examination, several direct smears of the biopsies were stained with May-Gru ¨nwald-Giemsa stain (Merck) and microscopic examination was carried out.For histopathology, biopsies from the nasal cavity and biopsy material from the orbital mass were routinely fixed in 10 % formalin for about 24 h.Tissue samples were embedded in paraffin wax and sectioned at 4 mm using routine methods.Staining for light microscopy was performed with haematoxylin and eosin (H&E) as well as periodic acid-Schiff (PAS) in an automated stainer (HMS740; Microm).

Microbiology and molecular analysis
Bacteriological cultivation was performed on sheep blood agar, Gassner agar and two anaerobic media: Zeissler agar (Columbia agar base with D-(+)-glucose at 10 g l -1 ) and Schaedler agar.The agar base was purchased from Merck and Becton Dickinson.For mycological cultivation, biopsy material was homogenized and streaked on modified Kimmig agar plates (15.0 g peptone l -1 , 1.0 g NaCl l -1 , 19.0 g D-(+)-glucose l -1 , 15.0 g agar agar l -1 , 5.0 ml glycerine/l, 50 mg Penicillin/l, 25 mg Streptomycin/l, pH 6.5) with and without cycloheximide (250 mg l -1 ), modified Dixon agar (36 g malt extract l -1 , 6 g peptone l -1 , 20 g ox gall l -1 , 10 ml Tween 40/l, 4 ml olive oil, 20 g agar agar l -1 , 950 ml dH 2 O) and brain-heart infusion agar (BHI) supplemented with 5% defibrinated sheep blood.Kimmig agar plates and Dixon agar plates were incubated for up to 26 days at 28 and 37 uC, whereas the BHI agar plates were incubated at 37 uC in CO 2 -enriched (10%) atmosphere.Throughout the incubation period, the colonies that grew were examined phenotypically at least once a week, and adhesive tape or needle preparations with lactophenol cotton blue or phloxine were prepared for microscopic examination.To examine the ability of the fungus to produce melanin, it was cultured at 28 and 37 uC on plates containing a lower concentration of glucose (5 g l 21 ).
For molecular analysis, DNA from the fungal colonies was isolated using hexadecyltrimethyl ammonium bromide (CTAB) (AppliChem) following a protocol slightly modified from Talbot (2001).In short, culture material of the fungus was homogenized and mixed with NaCl and CTAB lysis buffer.Several incubation and mixing steps were performed after addition of RNAse, 2-mercaptoethanol and chloroform/ isoamyl alcohol.After centrifugation at 10 000 g for 10 min, the aqueous phase was collected and fungal DNA was precipitated by adding isopropanol.The pellet was washed repeatedly and finally resuspended in Tris buffer to be used for PCR.Regions of interest were amplified using primers ITS1f (Gardes &Bruns, 1993) andITS4 (Innis, 1990) and KappaHifi polymerase (Peqlab) according to the protocol recommended by the manufacturer.Sequencing at GATC Biotech AG (Konstanz, Germany) and BiK-F (Biodiversity and Climate Research Centre, Frankfurt am Main, Germany) resulted in a consensus sequence (409 bp) of the ITS4 primer aligned with Geneious Pro 5.6.7 (Biomatters).For phylogenetic analysis the consensus sequence was aligned with ITS sequences published previously and listed in GenBank using MEGA6 (Tamura et al., 2013).Identification numbers and the origin of the sequences are given in Table 1.For distance analysis, a minimum-evolution tree (Tamura-Nei model) with partial deletion of gaps and 1000 bootstrap replications was reconstructed (Fig. S1, available in the online Supplementary Material).

Cytology
Cytological examination yielded a picture of a severe pyogranulomatous inflammation with severe hyperplasia of plasma cells and several yeast-like structures (Fig. 1a).

Histopathology
In both localizations, a marked pyogranulomatous and chronic suppurative inflammation was observed (Fig. 1b) and there was a suspicion of unstained fungal stages within the lesion.Using special stains, a moderate number of fungal organisms were visualized, characterized by intensely PAS-positive oval yeasts of 3-6 mm and small hyphae (Fig. 1c, d).

Bacteriological and mycological findings
Bacteriologically, only poor growth of Staphylococcus felis was diagnosed, whereas mycological examination discovered pure growth of small white colonies with a smooth and creamy surface already after 72 h incubation at 28 uC as well as at 37 uC.During further incubation at 28 uC, a white airy mycelial phase developed in the centre of the colonies (Fig. 2a, b) consisting of hyphae with conidiogenous cells and conidia lying in groups or in a sympodial order (Fig. 2c, d), whereas the smooth appearance remained visible in the peripheral region.Microscopically, hyphae as well as yeast-like organisms (Fig. 2e, f) could be found here despite the low incubation temperature.Colonies at 37 uC maintained their smooth character on BHI plates but got a wrinkled surface on Kimmig and Dixon agar (Fig. 2g, h).Microscopically, in these plates yeast-like structures dominated but were mixed with septate hyphae.Neither primarily nor after subcultivation or changing glucose concentration, was pigmentation observed.
ITS sequences of 44 Sporothrix species from different countries were compared using a minimum-evolution tree with 1000 bootstrap replications (Fig. S1).The sequence obtained from this isolate was placed at a relatively short distance from species isolated from environmental sources, which is consistent with previously analysed isolates in Germany (Zhou et al., 2013).Even though a strict separation between isolates from environmental versus clinical sources surrounding the sequence of this case was not evident, it seemed to be at a far distance from species of the complex building the clinical clade with higher pathogenic potential.

Treatment
The cat was treated initially with meloxicam (0.05 mg kg 21 per os, once a day) for 2 days.After that, itraconazole (5 mg kg 21 per os, once a day) was given daily for 5 months.

Outcome and follow-up
Control examinations were performed after 10 days, 15 days and 5 months.During this period, the thrombocytopenia persisted, with platelet counts ranging between 20000 and 55000 l 21 , whereas respiratory symptoms and nasal discharge improved remarkably after 3 weeks and disappeared completely after 6 weeks.The computed tomography scan revealed no further destruction of the nasal cavity.

Discussion
The cause of the respiratory symptoms of the cat was a chronic pyogranulomatous inflammation triggered by a mycotic process.Chronic nasal diseases in cats are quite common and -apart from a large number of cases where an aetiological agent cannot be found -are mostly related to neoplasia such as lymphoma or carcinoma, or to infectious agents such as feline herpesvirus 1, feline calicivirus, Chlamydophila sp., Mycobacterium sp. and Cryptococcus sp.(Allen et al., 1999;Demko & Cohn, 2007;Henderson et al., 2004;Reed & Gunn-Moore, 2012).In this case, besides a few colonies of Staphylococcus felis, no other bacteria were found and therefore a bacterial event could be excluded.Although the cat has been examined thoroughly, a definite cause for the persisting thrombocytopenia could not be found.Cytological examination of a bone-marrow aspirate showed an active bone marrow with megakaryocytes present.Taking into account the report by Miller & Lunn (2007) who stated that exam-inations of bone-marrow aspirates in a stage of severe isolated thrombocytopenia may not be of diagnostic or prognostic value, and due to the severity of the thrombocytopenia in the periphery, an immune-mediated process as a side effect of the mycosis had to be suspected.
Referring to the most prominent clinical signs (central impression of the nose, nasal discharge and a round mass near to the orbita), differential diagnoses such as neoplasia, mycobacteriosis, feline leprosy syndrome, nocardiosis and aspergillosis (Giordano et al., 2010) were considered.However, the massive destruction of the nasal cavity together with the cytological and histopathological finding of yeastlike organisms quickly raised suspicion of a dimorphic fungus, especially with regard to the relatively high sensitivity of cytopathological examination as a diagnostic feature (Pereira et al., 2011).Dimorphic fungi with a cutaneous manifestation in cats include Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioides immitis and S. schenckii (Blondin et al., 2007;Davies & Troy, 1996;Fischer et al., 2013;Greene & Troy, 1995).Besides their variable endemic regions, these fungi differ in the appearance of their yeastlike phase in the cytological and histopathological examination: Blastomyces dermatitidis yeasts consist of a thick wall, a small capsule and broad-based budding figures, Histoplasma capsulatum is often present in the form of round yeasts with a small capsule within macrophages, organisms of Coccidioides immitis present as large spherules and yeasts of S. schenckii are pleomorphic (Valenciano & Cowell, 2014).Differences also exist concerning their morphological appearance in culture and their clinical manifestation.Besides S. schenckii and Histoplasma capsulatum var.duboisii, all other mentioned dimorphic fungi predominantly infect the lungs and cause systemic disease (Hogan et al., 1996;Medleau et al., 2007;Moore et al., 2011).
Several phylogenetic studies have shown the heterogeneity of the genus Sporothrix and suggest that S. schenckii var.schenckii should not be the only member considered as the agent of sporotrichosis (Rodrigues et al., 2013a, b;Zhou et al., 2013).However, even though different species have been suggested, discussion of their level of virulence  was controversial as it seemed to vary between different species as well as within the same isolate according to its site of isolation (Arrillaga-Moncrieff et al., 2009;Brito et al., 2007;Howard & Orr, 1963).Melanin is considered a crucial virulence factor of S. schenckii isolates, although its expression varies among the species.For instance, Nobre et al. (2005) reported a difference between pigmented S. schenckii strains isolated from fixed cutaneous lesions versus systemic forms (Davis, 1915;Madrid et al., 2010b;Nobre et al., 2005).Comparing the morphology of the fungus isolated from this case with previously published cases of sporotrichosis in cats, its culture appearance of wrinkled to smooth colonies together with oval-shaped conidia growing in groups or in a sympodial manner is one of the main characteristics of the genus Sporothrix (de Hoog, 1995; Madrid et al., 2012).However, as it remained amelanotic throughout the whole incubation process regardless of different culture conditions (Almeida-Paes et al., 2009) and due to the lack of pigmentation in the histopathological examination, its classification outside the clinical clade of S. schenckii sensu stricto seems to be justified.The failure to produce melanin could be consistent with environmental strains like S. pallida.On the other hand, these strains are not usually responsible for major infections in immunocompetent patients, so it remains uncertain whether the strain in this case might have caused severe disease due to an immunocompromised status of the cat or, even though potentially related to environmental strains, it has a higher virulence in feline sporotrichosis than should be expected.Likewise, the simultaneous expression of yeast-like cells and hyphae might be due to the isolation site of the organism (Kwon-Chung, 1979).Clinical symptoms of feline sporotrichosis can vary and include cutaneous, lymphocutaneous and systemic forms (Schubach et al., 2004).Cutaneous lesions in particular are described as multiple, often-ulcerating nodules and are associated with a high fungal load (dos Santos et al., 2013;Miranda et al., 2013).Assuming a cutaneous form of feline sporotrichosis in this case, inoculation of conidia after a trauma of the skin seems to be rational.However, as the clinical picture presented was atypical in the sense of just one non-ulcerating cutaneous nodule, the possibility of air-borne transmission of conidia with manifestation in the nasal cavity should be considered.
As far as phylogenetic classification is concerned, the short ITS sequence in combination with relatively low bootstrap values did not enable a more specific nomenclature of the isolated fungus at this point.However, further molecular investigation, such as through examination of the calmodulin gene, remains to be performed so that a phylogenetic analysis with sequences especially from cases of feline sporotrichosis can be conducted (Marimon et al., 2006;Romeo et al., 2011).
As this fungus has been isolated from a clinical case and therapy with itraconazole dissolved the symptoms, it most likely represented an atypical case of feline sporotrichosis caused by Sporothrix sp.related to the environmental clade.Epidemiological evidence is difficult due to the lack of comparable cases in Germany.To the best of our knowledge, this case of feline sprotrichosis is the third described in Germany throughout the last 10 years and, although the infectious agent in the case in 2010 was described as a pigmented S. schenckii isolate, a zoonotic potential cannot be excluded (Weingart et al., 2010).This case report therefore once again emphasizes the fact that uncommon agents should be taken into account in chronic dermal infections in cats.

Fig. 2 .
Fig. 2. (a, b) Back (a) and front (b) sides of a colony of Sporothrix sp.grown at 28 8C for 26 days on modified Kimmig agar.(c, d) Conidiogenous cells with conidia in clusters (c) or arising directly from hyphae of Sporothrix sp.(d).(e, f) Oval to pyriform yeast-like structures of Sporothrix sp.grown at 28 8C on modified Kimmig agar.(g) Smooth colony morphology of Sporothrix sp.grown at 28 8C for 19 days on modified Kimmig agar.(h) Wrinkled colony morphology of Sporothrix sp.grown at 37 8C for 12 days on modified Kimmig agar.