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PubMed2001

Phenotypic characterization of Microsporum canis isolated from cats and dogs.

To characterize strains of Microsporum canis that infect dogs and cats in São Paulo city, 30 isolates of this dermatophyte were tested for their ability to assimilate carbon and nitrogen sources, for proteinase and phospholipase secretion, for susceptibility to yeast killer toxins, and for susceptibility to the antifungals fluconazole, ketoconazole, itraconazole, 5-fluorocytosine and amphotericin B, in E test. All samples assimilated the nitrogen sources asparagine, ammonium sulphate, urea and sodium nitrate, as well as the carbon sources inulin, mannitol, trehalose, meso-erythritol, maltose, mannose, sorbitol, cellobiose, fructose and dextrin. Not all the samples assimilated adonitol, galactose, arabinose, rhamnose, raffinose, melibiose, ribose and sucrose, and none of them was capable of growing with dulcitol, lactose, or xylose as the only carbon source. Proteinase and phospholipase secretion was observed for most isolates. In the test of yeast killer toxin, 10 types could be identified, with four types exclusively observed in isolates from dogs and two types exclusively observed in isolates from cats. In the E test, all isolates were found to be resistant to the fluconazole and 5-fluorocytosine, while they were variably sensitive to amphotericin B, ketoconazole and itraconazole. When the data were submitted to the qualitative analysis in the matrix distance program FITOPAC, the similarity of the isolates could be assessed.

Animals↗
PubMed1998

Antimicrobial effects of aqueous plant extracts on the fungi Microsporum canis and Trichophyton rubrum and on three bacterial species.

Aqueous extracts of 10 plants were tested for their ability to inhibit Trichophyton rubrum and Microsporum canis, the aetiological agents of dermal fungal infections in humans. These extracts were also evaluated for their activity against some bacteria. Aqueous extracts from the leaves of Inula viscosa produced detectable antifungal activity against these dermatophytes.

Anti-Bacterial Agents↗
PubMed2002

Terbinafine: efficacy and tolerability in young children with tinea capitis due to Microsporum canis.

AIMS: We carried out an open, prospective, uncontrolled study to evaluate the efficacy and tolerability of terbinafine in the treatment of young children with tinea capitis due to Microsporum canis. METHODS: A total of 83 healthy, immunocompetent children (age range 2-13 years) were enrolled in eight centres in Estonia, Latvia and Lithuania. Patients received oral terbinafine in dosages based on weight, 62.5 mg for those weighing 10-20 kg, 125 mg for 20-40 kg, plus application of topical 1% terbinafine cream twice daily to affected areas. Treatment lasted for 4 weeks, followed by an 8-week observation (treatment-free) period. All the subjects were assessed for efficacy and tolerability at 12 weeks. RESULTS: Eighty-one subjects were available for assessment at 12 weeks: 32 had completely recovered, with no evidence of relapse during the observation period, and 21 showed mycological cure, but presented residual physical signs of infection. Thus the effective cure rate was 65.4% in an infection known to be more difficult to cure than other causes of tinea capitis. Terbinafine was well tolerated by these children. CONCLUSIONS: This study showed effective cure of two-thirds of 81 cases of tinea capitis caused by M. canis with a 4-week course of treatment. As one-third of the cases did not respond to treatment, we suggest using combined oral and topical treatment with terbinafine in children with tinea capitis caused by M. canis.

Administration, Oral↗
PubMed2002

Isolation of a Microsporum canis gene family encoding three subtilisin-like proteases expressed in vivo.

Microsporum canis is the main agent of dermatophytosis in dogs and cats and is responsible for frequent zoonosis. The pathogenesis of the disease remains largely unknown, however. Among potential fungal virulence factors are secreted keratinolytic proteases, whose molecular characterization would be an important step towards the understanding of dermatophytic infection pathogenesis. M. canis secretes a 31.5 kDa keratinolytic subtilisin-like protease as the major component in a culture medium containing cat keratin as the sole nitrogen source. Using a probe corresponding to a gene's internal fragment, which was obtained by polymerase chain reaction, the entire gene encoding this protease named SUB3 was cloned from a M. canislambdaEMBL3 genomic library. Two closely related genes, termed SUB1 and SUB2, were also cloned from the library using as a probe the gene coding for Aspergillus fumigatus 33 kDa alkaline protease (ALP). Deduced amino acid sequence analysis revealed that SUB1, SUB2, and SUB3 are secreted proteases and show large regions of identity between themselves and with subtilisin-like proteases of other filamentous fungi. Interest ingly, mRNA of SUB1, SUB2, and SUB3 were detected by reverse transcriptase nested-polymerase chain reaction from hair of experimentally infected guinea pigs. These results show that SUB1, SUB2, and SUB3 encode a family of subtilisin-like proteases and strongly suggest that these proteases are produced by M. canis during the invasion of keratinized structures. This is the first report describing the isolation of a gene family encoding potential virulence-related factors in dermatophytes.

Amino Acid Sequence↗
PubMedDate not supplied

Tinea incognito due to Microsporum gypseum in three children.

Tinea incognito is a dermatophytosis of atypical clinical character due to the absence of classic features of ringworm. It is caused by prolonged use of topical steroids, sometimes prescribed as a result of incorrect diagnosis. The cases reported in the literature have different clinical presentations and have generally been in adults. We report three children with tinea incognito in whom the lesions were psoriasis-like, eczema-like, and lichenoid, respectively. Diagnosis was confirmed by mycologic examination, which led to the identification of Microsporum gypseum, a geophilic dermatophyte which is an infrequent agent of mycotic infection in humans.

Child↗
PubMedDate not supplied

Microsporum canis tinea capitis in an 8-month-old infant successfully treated with 2 weekly pulses of oral itraconazole.

We report an 8-month-old girl with tinea capitis caused by Microsporum canis which was successfully treated with two pulses of oral itraconazole 50 mg/day, each of 1 week duration with an interval of 2 weeks. M. canis tinea capitis can be a difficult therapeutic problem in an infant, as the treatment is usually long, lasting up to 4-6 weeks with griseofulvin and 6-12 weeks with terbinafine, and the desire to provide a safe systemic therapy is particularly important. With an intermittent regimen the duration of treatment with active drug can be much shorter compared to continuous therapy. We found that itraconazole pulse therapy for M. canis tinea capitis was a safe and effective treatment in an infant.

Administration, Oral↗
PubMedDate not supplied

Doubled dose of oral terbinafine is required for Microsporum canis tinea capitis.

Fourteen children, ages 1-15 years, with Microsporum canis tinea capitis were given oral terbinafine for 4 weeks at the recommended daily dose according to their weight: 10-20 kg, 62.5 mg; 20-40 kg, 125 mg; > 40 kg, 250 mg. Because none of the patients had clinically responded to treatment by week 4 the dose of terbinafine was doubled (up to 250 mg) for an additional 4-8 weeks in six patients, and continued at the original dose in six patients. Two patients dropped out of the study. On final evaluation, four patients were cured after 8-12 weeks of treatment. All cured patients received the doubled dose of terbinafine, except for one who was on the usual adult dose of 250 mg from the onset. Oral terbinafine was well tolerated by all but one patient, who experienced gastrointestinal disturbance and slightly raised transaminase levels during the first 4 weeks of treatment. Terbinafine side effects were not correlated with dosage or duration of treatment. We conclude that oral terbinafine should be dosed for M. canis tinea capitis at a revised schedule according to body weight: 10-25 kg, 125 mg/day and > 25 kg, 250 mg/day. The appropriate duration of terbinafine treatment remains to be determined.

Administration, Oral↗
PubMed2000

A study of the efficacy of topical and systemic therapy for the treatment of feline Microsporum canis infection.

Microsporum canis infection was induced in 21 healthy SPF-derived cats. Once infection was established (4 weeks after inoculation) the cats were divided into three equal groups housed in separate rooms and monitored for 16 weeks. During this time, group A cats received oral griseofulvin at approximately 50 mg/kg daily and were shampooed twice weekly with a product containing chlorhexidine and miconazole. Group B cats were treated with griseofulvin alone, and group C cats served as untreated controls. The cats were examined on a weekly basis and the severity of lesions was scored semi-quantitatively. In addition, hair samples were collected from each cat on a weekly basis by the MacKenzie brush technique and by the sticky-tape method. A semi-quantitative scoring system was also used for the assessment of fungal (M canis) growth. Generally, significant differences in clinical scores were not seen between the groups although at weeks 3, 4 and 11 there was a significant difference (P< or =0.015) with cats in group A having significantly lower median scores than those in group C. Median times to clinical resolution (return of clinical scores to zero) in groups A, B and C were at treatment weeks 2, 9 and 12, respectively (P>0.05). Median times for mycological resolution (persistently negative culture results) for groups A, B and C were at treatment weeks 2, 9 and 12, respectively, for the MacKenzie brush technique and at weeks 4, 8 and 12 for the sticky-tape technique. For both these results, the groups differed significantly (P< or =0.001) and in both instances group A had significantly more rapid resolution than groups B or C. Median culture scores were significantly different between the three groups using one or both of the sampling techniques at week 2 through to week 12 of treatment with median scores for either group A alone, or groups A and B being significantly lower than group C (P< or =0.026). These results showed a benefit from the addition of twice-weekly chlorhexidine-miconazole shampooing to systemic griseofulvin therapy alone in the treatment of M canis infected cats.

Administration, Topical↗
PubMed1990

The value of electrophoretic protein patterns for the study of Microsporum canis.

Whole-cell protein analysis of clinical isolates of Microsporum canis by polyacrylamide gel electrophoresis (PAGE) showed small but inconsistent differences in protein patterns. All clinical isolates closely resembled the (-) mating type of Arthroderma otae. A reference strain of A. otae (+) mating type from Japan gave a PAGE pattern which was distinct from those of the clinical isolates. Mating studies showed that the progeny of these reference (+) and (-) mating types had a variety of distinct protein patterns. Protein patterns can therefore distinguish between genetically different strains of M. canis (A. otae) suggesting that clinical isolates have a common clonal origin.

Animals↗
PubMed1991

Studies on the mode of action of tolnaftate in Microsporum gypseum.

Studies were performed on the mode of action of tolnaftate and resistance to this drug in Microsporum gypseum. Cells grown in the presence of tolnaftate (at the IC 50) showed a reduced content of total phospholipids and sterols whereas there was an increase in total RNA content. Incubation of cells with tolnaftate (at 10 x MIC), followed by addition of different macromolecule precursors revealed inhibition of the biosynthesis of all macromolecules except for RNA. The activity of membrane-bound enzymes did not change on treatment with tolnaftate (10 x MIC) whereas an increase in the leakage of intracellular 32P was observed. The content of total phospholipids was higher in tolnaftate-resistant cells, whereas the content of total sterols, DNA, RNA and protein was comparable to that of susceptible cultures. Activity of phosphodiesterase decreased and 5'-nucleotidase increased in tolnaftate-resistant cells. Our results suggest that the antifungal activity of tolnaftate is due to differential action on various targets site(s) which are modified in strains resistant to the drug.

DNA, Fungal↗
PubMed2003

Humoral and cellular immune response to a Microsporum canis recombinant keratinolytic metalloprotease (r-MEP3) in experimentally infected guinea pigs.

In order to better understand the host-fungus relationship in Microsporum canis dermatophytosis and to identify major fungal antigens, the immune response to a crude exoantigen preparation and to a purified recombinant keratinolytic metalloprotease (r-MEP3) was evaluated in guinea pigs experimentally infected with M. canis. Humoral and cellular immune responses were assessed from day 0 to day 57 post-infection (PI), the former by enzyme-linked immunosorbent assay (ELISA) and the latter via a lymphocyte proliferation assay. Infected guinea pigs developed humoral and cellular responses to both M. canis exoantigen and r-MEP3, while no specific immune response to these antigens was observed in control animals. This is the first report on the development of both humoral and cell-mediated immune responses to a purified keratinase in M. canis dermatophytosis.

Animal Experimentation↗
PubMed2004

Evaluation of Microsporum canis in different methods of storage.

The main objective of this investigation was to evaluate different methods of storage for Microsporum canis based on materials and equipment that are readily available in developing countries. We tested 32 strains of M. canis at - 20 degrees C in potato dextrose agar (PDA) in its plain condition, or amended with 10% dimethyl sulfoxide or with 10% glycerol. In addition, we tested 25 degrees C storage of isolates in plain saline (0.9% NaCl) and in saline covered with a mineral-oil layer. After 9 months of storage, none of the M. canis strains frozen in PDA supplemented with glycerol survived, while only 16 and 6%, respectively, of the isolates in plain and DMSO medium lost viability. Nine month's storage in saline with or without mineral oil increased the amount of pleomorphic development of sterile hyphae; this phenomenon occurred at a significantly higher level than was seen in isolates stored at -20 degrees C. The physiological characteristics of M. canis were not affected by the different storage tests. The results suggest that, in order to ensure optimal viability, purity and pristine isolate condition, each M. canis isolate maintained should be held in at least two methods of storage, namely, PDA at -20 degrees C and saline with a mineral-oil layer at 25 degrees C.

Animals↗
PubMed2005

Reproducible genetic transformation system for two dermatophytes, Microsporum canis and Trichophyton mentagrophytes.

A reproducible genetic transformation system was developed for two major dermatophytes, Microsporum canis and Trichophyton mentagrophytes. Two circular transformation vectors carrying either the bacterial hygromycin B phosphotransferase gene (hph) or both the hph and green fluorescent protein (eGFP) genes under the control of a promoter sequence from Cochlibolus heterostrophus were introduced independently into the protoplasts by a polyethylene glycol (PEG)-mediated method. Polymerase chain reaction (PCR) showed that the hph gene was integrated randomly into the chromosomal DNA of the transformants through non-homologous recombination. Southern blotting analysis also demonstrated a single or multiple integration of the hph gene into the chromosomal DNA. Fluorescence due to eGFP gene expression was observed in the T. mentagrophytes transformants, and the transformants retained mitotic stability through subculture. This reproducible transformation system provides a method for the genetic manipulation of these pathogens, which will facilitate detailed molecular analysis of dermatophytosis.

Blotting, Southern↗
PubMed2006

Isolation, characterization, and disruption of dnr1, the areA/nit-2-like nitrogen regulatory gene of the zoophilic dermatophyte, Microsporum canis.

A homolog of the major nitrogen regulatory genes areA from Aspergillus nidulans and nit-2 from Neurospora crassa was isolated from the zoophilic dermatophyte, Microsporum canis. This gene, dnr1, encodes a polypeptide of 761 amino acid residues containing a single zinc-finger DNA-binding domain, which is almost identical in amino acid sequence to the zinc-finger domains of AREA and NIT-2. The functional equivalence of dnr1 to areA was demonstrated by complementation of an areA loss-of-function mutant of A. nidulans with dnr1 cDNA. To further characterize this gene, dnr1 was disrupted by gene replacement based on homologous recombination. Of 100 transformants analyzed, two showed the results expected for replacement of dnr1. The growth properties of the two dnr1(-) mutant strains on various nitrogen sources were examined. Unlike the A. nidulansareA(-) mutant, these dnr1(-) mutants showed significantly reduced growth on ammonia, a preferred nitrogen source for fungi. These mutant strains were also able to utilize various amino acids for growth. In comparison with wild-type M. canis, the two dnr1(-) mutants showed reduced growth on medium containing keratin as the sole nitrogen source. This is the first report describing successful production of targeted gene-disrupted mutants by homologous recombination and their phenotypic analysis in dermatophytes.

Amino Acid Sequence↗
PubMed2000

Molecular and conventional taxonomy of the Microsporum canis complex.

The validity of taxa around Microsporum canis was evaluated by a combination of phenetic and molecular methods. Morphological and physiological features were compared with results of sequencing of the internal transcribed spacer (ITS) region of the ribosomal operon, polymerase chain reaction (PCR) fingerprinting and amplified fragment length polymorphism (AFLP) analysis. The seven species investigated seem to be infraspecific taxa and were reclassified or synonymized as M. canis (teleomorph: Arthroderma otae), M. ferrugineum, and M. audouinii.

Animals↗
PubMed2001

A specific PCR assay for the dermatophyte fungus Microsporum canis.

A DNA fragment of approximately 1.2 kb, generated from the common dermatophyte Microsporum canis by arbitrarily primed polymerase chain reaction (PCR) using random primer OPU13, was cloned and sequenced. Based on the resulting sequencing data, a forward primer (MC1F) and a reverse primer (MC1R) have been designed and assessed by PCR for their usefulness in the improved identification of M. canis. The results obtained suggest that these primers are specific for M. canis, as a band of 900 bp was amplified in PCR with genomic DNA from M. canis only, and not from any of the other dermatophyte species or varieties, other fungi or common bacteria examined. Combining this PCR technique with a rapid mini-preparation method for fungal DNA, a definitive diagnosis of M. canis can be achieved within a day from the primary cultures. Future refinement of a DNA purification protocol from clinical specimens would further enhance the potential of the PCR based test for improved detection and identification of M. canis.

DNA, Fungal↗
PubMed2001

A case of feline otitis externa due to Microsporum canis.

We describe a case of a 3-month-old Persian male cat with a persistent waxy, ceruminous, otic discharge in both external auditory canals. After microbiological culture, only Microsporum canis was isolated with no concurrent fungal or bacterial flora. Fungal otitis due to dermatophytes was diagnosed and specific treatment with griseofulvin was administered. After 4 weeks of treatment, no M. canis colonies were isolated and clinical recovery had occurred. In view of this case, dermatophytosis should be included in the differential diagnosis of feline otic conditions, particularly when otitis becomes chronic or is refractory to treatment.

Animals↗
PubMed2001

Purification and characterization of a 43.5 kDa keratinolytic metalloprotease from Microsporum canis.

A keratinolytic protease secreted by a feline clinical isolate of Microsporum canis cultivated in a broth containing feline keratin as the sole nitrogen source was purified from the culture filtrate by affinity chromatography on bacitracin-agarose and by hydrophobic chromatography on octyl-agarose. The enzyme had an apparent molecular mass of 43.5 kDa and the pI was 7.7. It had a significant activity against keratin azure, elastin-Congo red and denatured type I collagen (azocoll). Using the latter substrate, the optimum pH was around 8 and the apparent optimum temperature around 50 degrees C. The protease was strongly inhibited by 1,10-phenanthroline, phosphoramidon and EDTA. The first 13 N-terminal amino acid sequence showed a 61% homology with that of the extracellular metalloprotease of Aspergillus fumigatus and with the neutral protease I of A. oryzae, confirming that this 43.5 kDa keratinase is a metalloprotease. This keratinolytic metalloprotease could be a virulence-related factor involved in pathophysiological mechanisms of M. canis dermatophytosis.

Amino Acid Sequence↗