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PubMed1995

Identification, localization and possible role of calmodulin like protein in phospholipid synthesis of Microsporum gypseum.

Calmodulin like protein has been identified for the first time in dermatophyte--M. gypseum (by specific radioimmunoassay). Maximum amount of this protein was present in the early and mid log phase of growth and was mainly localized in the cytosolic fraction. Cells treated or grown with calmodulin antagonists (phenothiazine and trifluoperazine) exhibited lower uptake of [14C]acetate or labelled phosphate into phospholipids. This is probably due to lower levels of calmodulin seen in these cells. Our results suggest the relationship between calmodulin levels and phospholipid synthesis in Microsporum gypseum.

Calmodulin↗
PubMed1994

Possible role of calcium in phospholipid synthesis of Microsporum gypseum.

The effect of calcium on lipid synthesis in Microsporum gypseum was examined by growing these cells in calcium supplemented (1 mM to 10 mM) medium. Maximum incorporation of [14C]acetate into total lipids and phospholipids was observed in cells grown with 6 mM calcium. This was probably due to a 3-fold increase in total calcium levels as incorporation of label was inhibited in total lipids (33%) and phospholipids (20.5%) in calcium-grown cells which were preincubated with the calcium specific chelator ethylene glycolbis (beta-aminoethyl ester) N,N,N',N',-tetracetate (EGTA). Increased incorporation of [14C]acetate into phospholipids was further supported by increase in the activity of key phospholipid biosynthetic enzymes (glycerolkinase and glycerol-3-phosphate acyltransferase) as well as the increase in phospholipid content in calcium-grown cells, which suggests a correlation between increased calcium levels and phospholipid biosynthesis in M. gypseum.

Acetates↗
PubMed1986

Mycetoma-like granuloma in a cat caused by Microsporum canis.

A solitary subcutaneous nodule from a Persian cat consisted of coalescing mycetoma-like granulomas containing abnormal hyphae of Microsporum canis. Overlying hair follicles were heavily laden with dermatophytes but had little associated perifollicular inflammation. The complex classification of dermatophyte-induced dermal inflammatory lesions is reviewed.

Animals↗
PubMed1995

Investigations of a killed dermatophyte cell-wall vaccine against infection with Microsporum canis in cats.

A laboratory-prepared killed Microsporum canis cell-wall vaccine was evaluated under conditions simulating an accidental infection of a cattery, by inoculating eight- to nine-week-old cats with the vaccine or with a placebo control. The vaccinated cats developed high titres of anti-dermatophyte IgG as measured by an ELISA, and a small cell-mediated response against M canis as measured by a lymphocyte blastogenesis assay, using a whole fungus extract. After being inoculated the cats were challenged by the introduction of an infected cat into the same room. All the vaccinated and control cats became culture-positive for M canis within four weeks of the introduction of the infected cat. Four of the six control cats and all the vaccinated cats developed lesions consistent with dermatophytosis within 16 weeks after exposure to the infected cat.

Animals↗
PubMed1994

Immunogenicity in guinea-pigs of a crude ribosomal fraction from Microsporum canis.

The immunogenicity of a crude ribosomal fraction (CRF) extracted from Microsporum canis was tested by assessing protection of vaccinated guinea-pigs (GP) against a challenge with the dermatophyte. Stimulation of the humoral as well as the cellular immune systems of these animals was evaluated by ELISA and the lymphocyte stimulation tests, respectively. In addition, the immune response elicited by the M. canis CRF was examined for cross-reactivity towards Trichophyton verrucosum antigen. The duration of the infection induced by M. canis in GP was reduced to 8 days in vaccinated animals versus 37 days in the control GP. Both humoral and cellular immune systems were stimulated by the CRF. Sera of GP vaccinated with M. canis CRF revealed presence of anti-T. verrucosum antibodies, albeit at titres significantly lower than against M. canis.

Animals↗
PubMed1994

Development of an experimental model of Microsporum canis infection in cats.

An experimental infection model was developed for reliable induction of Microsporum canis skin infections in cats, using a defined number of macroconidia harvested from the fungus in culture. The strain of M. canis used produced highly fluorescent hairs under ultraviolet illumination. Kittens 8 to 9 weeks of age (n = 6) received 10(5) macroconidia applied topically to a closely-shaved area of skin. Sites were dressed with an occlusive bandage for 3 days, then grooming was restricted for an additional 4 weeks. Lesions were first observed 2 weeks after inoculation, enlarged over the following 6 to 8 weeks, then decreased in size and appeared healed at 12 to 14 weeks after inoculation. Cats often developed satellite lesions on the face, ears, or other body regions. The experimental infections strongly resembled moderately severe cases of naturally-occurring feline dermatophytosis in clinical patients. This experimental infection model will be useful for evaluation of topical and systemic treatments for feline M. canis infection.

Animals↗
PubMed1995

Production and use of monoclonal antibodies to Microsporum canis.

Microsporum canis NCPF 179 and M. canis NCPF 177 dermatophyte cytoplasmic extracts (DCEs) were used as antigens to generate monoclonal antibodies (mAbs). Hybridomas with supernatants of optical density (OD) > 1 for homologous dermatophyte cytoplasmic extracts (CE) and OD < 0.5 for heterologous CE of Candida albicans tested by the enzyme-linked immunosorbent assays (ELISA) were selected for cloning. mAbs secreted by cloned hybridoma lines were screened against CE of M. canis, M. gypseum, M. equinum, M. distortum, Trichophyton verrucosum, C. albicans, Malassezia pachydermatis and Aspergillus fumigatus. The ELISA performed on clone supernatants identified a variety of different activities between the dermatophyte and other fungal CEs. The selected mAbs were used for immunoblotting of native and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Immunoblots with some of the mAbs tested allowed the differentiation of strains belonging to different dermatophyte species and isolates belonging to the M. canis.

Animals↗
PubMed1990

Detection of keratinolytic proteinase in skin tissues from guinea pigs infected with Microsporum canis by an immunoperoxidase technique.

An immunoperoxidase technique was performed to detect keratinolytic proteinase (KPase) in sections of the skin taken from guinea pigs infected with Microsporum canis and in cultured M. canis using polyclonal antisera to purified KPase. Of tissue samples from guinea pigs infected with M. canis, all sections of erythematous lesions showed positive staining mainly in the horny layer and the hair follicles. Positive depositions were seen only at the level of the outer and inner root sheaths of the hair follicles in sections of skin lesions showing scales and crusts. However, sections from areas of alopecia following desquamation of the crust showed no depositions of bright red reaction products. The patterns of deposition of KPase according to the clinical course of experimental dermatophytosis were consistent with the existence of organisms observed by PAS and methenamine silver stains. These results suggest that KPase may be produced during infection with M. canis after the development of erythema and before desquamation of the crust.

Animals↗
PubMed2005

Fluconazole upregulates sconC expression and inhibits sulphur metabolism in Microsporum canis.

Azole derivatives such as fluconazole are the mainstay of therapeutic agents for the treatment of fungal infections. Their mode of action involving alteration in the conversion of lanosterol to ergosterol is well established. Here we report the effect of fluconazole on the sulphur metabolism negative regulator gene (sconC) in Microsporum canis. Characterization of the M. canis sconC gene revealed that its ORF is comprised of 495bp interrupted by four introns of 47-70bp. Exposure of M. canis in suspension to fluconazole upregulates sconC mRNA level and protein expression as determined by Northern and Western blot analysis, respectively. Upregulation of sconC was accompanied by inhibition of sulphur metabolism of the fungus resulting in a greatly reduced incorporation of radioactive labelled sulphuric acid into fungal proteins. These data establish that in addition to its action on ergosterol synthesis, fluconazole acts on other biological pathways in fungal cells.

Amino Acid Sequence↗
PubMed2005

Inter-single-sequence-repeat-PCR typing as a new tool for identification of Microsporum canis strains.

BACKGROUND: Microsporum canis is a ubiquitous dermatophyte that commonly causes human infections. Since contact with infected animals is the usual way of infection, tracing its source is an essential preventive measure. OBJECTIVE: To type isolates of M. canis from human patients whose skin was affected, and from some animals (dogs and cats) that were closely associated to the patients. METHODS: The inter-single-sequence-repeat-PCR (ISSR-PCR) technique has been used for typing 24 isolates of M. canis. Seventeen isolates tested were from human patients, 5 from cats and 1 from a dog RESULTS: A total of 21 genotypes were identified. The same genotype was found infecting a patient and a cat that was living closely with him, but another member of the same family proved to be infected with two genotypes different from that. Clinical specimens from two patients had been contaminated with the same genotype, probably in the laboratory where the samples were handled. CONCLUSION: These results demonstrate that ISSR-PCR polymorphism is a reliable method for the identification of the M. canis strains.

Animals↗
PubMed2002

Fluconazole downregulates metallothionein expression and increases copper cytotoxicity in Microsporum canis.

Azole antifungals are widely used to treat infections with dermatophyte fungi. Whereas it is well established that this class of drugs interferes with fungal ergosterol synthesis, little is known about its potential other biological effects. Here we report the isolation and structural organization of Microsporum canis metallothionein gene and demonstrate that fluconazole is able to downregulate the baseline as well as copper-induced expression of this gene. Since this effect occurred within 30 min after exposure of the fungus to fluconazole, it is unlikely that it is due to impaired ergosterol synthesis. Our additional demonstration that fluconazole enhances copper toxicity for M. canis suggests that inhibition of metallothionein expression by fluconazole is biologically relevant and may represent an important additional mode of the antifungal action of this drug. Therefore our data indicate that antifungal effects of azole derivatives might not only be due to interference with cell wall synthesis but may also affect other biological circuits within the fungal cells.

Amino Acid Sequence↗
PubMed1996

Acquired immunity in experimental feline Microsporum canis infection.

Seven adult cats were inoculated cutaneously at a shaved site with Microsporum canis; five (group A) had spontaneously recovered from a previous M canis infection, and two (group B) were dermatophyte-naive. Both of the dermatophyte-naive cats developed typical clinical disease, the lesions reaching maximal size four to five weeks after exposure, and resolving by week 13. Only one of the group A cats developed clinical disease; the lesions were more inflammatory in nature and of shorter duration and had resolved by day 25 after exposure. Blood samples collected from the seven cats before and four weeks after exposure were assessed for M canis-specific cellular (by lymphocyte proliferation assays) and humoral (by IgG and IgM ELISA) immune responses. After four weeks the group A cats had significantly (P < 0.05) increased cellular and IgG-humoral immune responses, and the group B cats also had increased cellular, and IgG and IgM responses but the increases were not significant.

Animals↗
PubMed1986

Microsporum nanum infection in hog farmers.

Microsporum nanum is a dermatophyte that can cause disease in both man and animals and is most frequently associated with ringworm infection in pigs. Human infections are rare in the literature. Three new cases of human M. nanum infection are reported here. Two patients had inflammatory tinea faciei and one had tinea cruris. The patients were successfully treated with clotrimazole or miconazole cream, sometimes combined with griseofulvin. All three patients were hog farmers and lived in the same small rural area. Occupational exposure is suggested as the cause of infection.

Adult↗
PubMed1987

The infected hairs of tinea capitis due to Microsporum canis: demonstration of uniqueness of the hair cuticle by scanning electron microscopy.

Scanning and transmission electron microscopic studies were done on scalp hairs of four patients infected with ectothrix Microsporum canis. Both freeze fracture and cross-sectioning of hairs revealed a thick sheath of fungal spores encircling the hair shaft beneath an intact cuticle. These spores were not visible on surface inspection but became apparent only where the cuticle had been rubbed off or broken. Daily selenium sulfide shampoos removed all of the spores from these sites. The cuticle is viewed as being an effective barrier to the penetration of fungi, so that the hair is vulnerable to fungus infection only deep within the hair follicle below the level of the mature cuticle. Once the fungus enters the hair cortex just above the hair bulb, it produces myriads of spores that remain trapped and hidden beneath the cuticle for the length of the intact hair.

Child↗
PubMed2000

Purification and characterization of cAMP dependent protein kinase from Microsporum gypseum.

A cyclic AMP dependent protein kinase (PKA), its regulatory (R) and catalytic (C) subunits were purified to homogeneity from soluble extract of Microsporum gypseum. Purified enzyme showed a final specific activity of 277.9 nmol phosphate transferred min(-1) mg protein(-1) with kemptide as substrate. The enzyme preparation showed two bands with molecular masses of 76 kDa and 45 kDa on sodium dodecyl polyacrylamide gel electrophoresis. The 76 kDa subunit was found to be the regulatory (R) subunit of PKA holoenzyme as determined by its immunoreactivity and the isoelectric point of this subunit was 3.98. The 45 kDa subunit was found to be the catalytic (C) subunit by its immunoreactivity and phosphotransferase activity. Gel filtration using Sepharose CL-6B revealed the molecular mass of PKA holoenzyme to be 240 kDa, compatible with its tetrameric structure, consisting of two regulatory subunits (76 kDa) and two catalytic subunits (45 kDa). The specificity of enzyme towards protein acceptors in decreasing order of phosphorylation was found to be kemptide, casein, syntide and histone IIs. Purified enzyme had apparent K(m) values of 71 microM and 25 microM for ATP and kemptide, respectively. Phosphorylation was strongly inhibited by mammalian PKA inhibitor (PKI) but not by inhibitors of other protein kinases. The PKA showed maximum activity at pH 7.0 and enzyme activity was inhibited in the presence of N-ethylmaleimide (NEM) which shows the involvement of sulfhydryl groups for the activity of PKA. PKA phosphorylated a number of endogenous proteins suggesting the multifunctional role of cAMP dependent protein kinase in M. gypseum. Further work is under progress to identify the natural substrates of this enzyme through which it may regulate the enzymes involved in phospholipid metabolism.

Cyclic AMP-Dependent Protein Kinases↗
PubMed2001

Chitin synthase 1 (Chs1) gene sequences of Microsporum equinum and Trichophyton equinum.

Chitin synthase 1 (Chs1) genes from Microsporum equinum and Trichophyton equinum were compared with those of the other dermatophytes. The Chs1 nucleotide sequences of these dermatophytes from horses showed more than 80% similarity to those of Arthroderma benhamiae, A. fulvum, A. grubyi, A. gypseum, A. incruvatum, A. otae, A. simii, A. vanbreuseghemii, Epidermophyton floccosum, T. mentagrophytes var. interdigitale (T. interdigitale), T. rubrum and T. violaceum. Especially high degree of nucleotide sequence similarity of more than 99% was noted between the Chs1 gene fragments of M. equinum and A. otae, and those of T. equinum, T. interdigitale and A. vanbreuseghemii, respectively. The phylogenetic analysis of their sequences revealed that M. equinum was genetically very close to A. otae and T. equinum to A. vanbreuseghemii. A molecular analysis of Chs1 genes will provide useful information for the genetic relatedness of M. equinum and T. equinum and confirm the value of DNA sequencing in identification of these two dermatophytes.

Animals↗
PubMed2001

Terbinafine hydrochloride treatment of Microsporum canis experimentally-induced ringworm in cats.

Cats represent the most important source of Microsporum canis infection to people. Terbinafine hydrochloride is commonly used in the treatment of microsporosis. Its fungicidal action permits short period of treatment. It was our objective to evaluate the effectiveness of this drug in treatment of microsporosis in cats. We treated nine experimentally M. canis infected cats with terbinafine at a dose of 10-20mg/kg SID (low-dose group, LDG), nine cats with 30-40mg/kg SID (high-dose group, HDG), and nine cats were left untreated (control group, CG). The drug's levels in cats' plasma and hair were measured by a reversed-phase high performance liquid chromatographic method (RP-HPLC) and the cats' cure was followed by Wood's lamp illumination, microscopic exam and fungal culture. We showed no difference between the clinical course in CG and LDG, but HDG were significantly differentiated from both other groups. Terbinafine levels in plasma at 120 days of treatment were not statistically different among LDG (4.13 microg/l) and HDG (5.48 microg/l), but levels in hair of LDG (1.24 microg/l) and HDG (3.62 microg/l) were significantly different. Terbinafine can be used for the treatment of microsporosis in cats in the dose of 30-40mg/kg SID.

Animals↗
PubMed2003

Recombinant expression and antigenic properties of a 31.5-kDa keratinolytic subtilisin-like serine protease from Microsporum canis.

A secreted 31.5-kDa keratinolytic subtilase (SUB3; AJ431180) is thought to be a Microsporum canis virulence factor and represents a candidate for vaccination trials. In this study, the recombinant keratinase (r-SUB3) was produced by the Pichia pastoris expression system and purified to homogeneity. Recombinant SUB3 displayed identical biochemical properties with the native protease. Experimentally cutaneously infected guinea pigs showed specific lymphoproliferative response towards r-SUB3, while no specific humoral immune response was induced except for one animal. The heterologous expression of SUB3 provides a valuable tool for addressing further investigations on the role of this keratinase in the specific cellular immune response and on its use in vaccination trials in the cat.

Animals↗