Onychomycosis caused by Malassezia pachydermatis in a dog

Background: Malassezia species are commensal skin organisms of warm-blooded vertebrates that can act as opportunistic pathogens. Malassezia pachydermatis is of importance in both veterinary and human medicine. Recognised initially as causes of infection of the skin, they are now known to be superficial commensals as well as potential causes of infections in domestic animals and more serious human conditions such as fungemia. They have also been implicated in the pathogenesis of allergic and other inflammatory diseases. Onychomycosis is defined as fungal infection of the claw (nail disease) caused by dermatophytes (Microsporum and Trichophyton genus), non-dermatophyte molds (hyaline and dematiaceous) and/or yeasts. The objective of this work was to report a case of canine onychomycosis by Malassezia pachydermatis based on reference standard diagnostics of this pathology. Case: A 9-year-old male West Highland White Terrier presented history of claw abnormality: brown staining and partly brittle. The dog has no apparent history of trauma on the affected claw and no skin disease was reported. Direct examination of claw was performed using the tape strip technique revealed Malassezia organisms. Nail fragments were cultured onto Sabouraud Dextrose Agar with Chloramphenicol and Cycloheximide. After 10 days of incubation (32ºC) nail fragments allowed the isolation of pure colonies of Malassezia pachydermatis. The histopathological evaluation was performed by Sector of Veterinary Clinical Pathology, Faculdade de Veterinária (FAVET/UFRGS) and fungal infection due to Malassezia pachydermatis was confirmed by histopathologic examination (Hematoxylin and eosin and Grocott’s methenamine silver stain) of the nail, that revealed abundant yeasts (blastoconidia). Discussion: Fungal infection of the skin, hair and nails are common and are primary caused by the dermatophyte molds. Non-dermatophyte molds isolated from nails constitute a long list, but only a few species cause onychomycoses. These include Scopulariopsis brevicaulis, Fusarium sp., Acremonium spp., Aspergillus spp., Penicillium spp. Yeasts like Malassezia and Candida spp. usually are saprobics and usually represent incidental contaminants associated with a non-sterile specimen at mycological culture, however in some cases they may parasite the nails and become an etiological agent of the disease. M. pachydermatis, normally present on the skin and in the ear canal of dogs and cats, can act as opportunistic pathogenic microbe and frequently causes dermatitis and otitis in mammals. In the case of onychomycosis, there is little evidence that Malassezia yeasts are implicated in nail plate invasion, although this may be a rare occurrence. All species of the genus are obligatory lipid-dependent forms, with the exception of M. pachydermatis, as well as absence of keratinolytic ability. Malassezia organisms were commonly retrieved from the subungual claw fold region of normal healthy dogs (should be considered resident microflora of the normal canine claw fold). Some authors suggests that high numbers of yeast can be detected on cytological evaluation of the skin surface of the canine claw fold from allergic dogs in both the absence and presence of concurrent signs of pruritus and paronychia

Susceptibilidade antimicrobiana de estafilococos, isolados de mastite bovina na zona da mata de Minas Gerais

Three hundred and fifty-four antibiograms were performed with 300 strains of Staphylococcus aureus and 54 of S. epidermidis using the diffusion method. Among the S. aureus, 165 (55%) were completely susceptible to the eleven antimicrobial agents tested; -67 (22,3%) resistants to penicillin G (PEN), 5 (1,7%) to tetracycline (TET), 4 (1,3%) to chloramphenicol (CLR), 2 (0,6%) to streptomycin and 2 (0,6%) to lincomycin in vitro. Multiple resistance was found in about 15% of the isolates, being most common the combinations involving PEN: STR: TET and CLR. According to the source of staphylococcal infection, 259 (86,3%) were strains of bovine origin, 29 (9,7%) human and 12(4%) canine, being the bovine ones much more resistant except to novobiocin, cloxacillin and rifamycin. Regarding the 54 S. epidermidis, 28 (51,8%) showed total susceptibility and 16 (29,6%) multiple resistance, specially combinations of PEN: STR: TET: CLR and Sulfisoxazole (SUL). All 354 strains were susceptible to rifamycin (rifampim).Foram realizados 354 antibiogramas com 300 estirpes de Staphylococcus aureus e 54 de S. epidermidis, através do método de difusão. Das 300 estirpes de S. aureus, 165 (55%) eram totalmente susceptíveis aos onze agentes antimicrobianos testados; 67 (22,3%) resistentes à penicilina G (PEN); cinco (1,7%) à tetraciclina (TET); quatro (1,3%) ao cloranfenicol (CLR); duas (0,6%) à estreptomicina (STR) e duas (0,6%) à lincomicina (LIN) in vitro. Resistência múltipla foi constatada em 14,9% das amostras, sendo mais comuns as combinações envolvendo PEN: STR: TET. CLR. De acordo com a fonte de infecção estafilocócica, 259 (86,3%) eram amostras de origem bovina, 29 (9,7%) de origem humana e 12 (4%) de origem canina, sendo as estirpes bovinas bem mais, resistentes, exceto para novobiocina, cloxacilina e rifamicina. Em relação às 54 estirpes de S. epidermidis, 28 (51,8%) apresentaram susceptibilidade total e 16 (29,6%), resistência múltipla, especialmente combinações de PEN: STR.TET.CLR e sulfisoxazole (SUL). Todas as 354 estirpes testadas foram susceptíveis à rifampicina

Canine Ringworm Caused by Trichophyton mentagrophytes - Detection by SYBR-Green real-time PCR

Background: Dermatophytes, fungi of universal distribution, invade semi or fully keratinized structures, such as skin, fur/hair and nails. The various species of dermatophytes are classified into three genera anamorphic: Microsporum, Trichophyton and Epidermophyton. The genus Epidermophyton includes only E. floccosum, that rarely affects animals. The main species responsible for the disease in dogs and cats are Microsporum canis, M. gypseum and Trichophyton mentagrophytes, which were characterized through conventional mycological methodology (microscopic examination with KOH and culture). Molecular methodologies, such as real-time PCR, can contribute to a rapid laboratory diagnosis, helping clinicians to initiate an early antifungal treatment. This case report describes a case of canine dermatophytosis due to Trichophyton mentagrophytes detected from a clinical sample by SYBR-Green real-time PCR.Case: A 8-year-old dog, rescued from the street, was referred to a private veterinary clinic in the city of Canoas, RS, Brazil, presenting generalized lymphadenomegaly, crusted lesions all over the body, generalized alopecia, signs of excoriation and epistaxis. Initially, were administered prednisone [1 mg/kg every 48 h, BID] and cephalexin [30 mg/kg, BID]. Weekly baths with benzoyl peroxide were also given. The therapy was not clinically successful. Wood’s Lamp Test was negative. As a differential diagnosis, PCR for detection of Leishmania was negative. Complete blood count and serum biochemical assay were also performed. For mycological diagnosis, hair specimen was clarified and examined microscopically using 10% potassium hydroxide (KOH) for the visualization of chains of arthroconidia (ectothrix invasion of hair). The infected hair was plated onto MycoselTM Agar, incubated at 28°C for 15 days. Microscopy of hyphae/ conidia and macroscopic colony characteristics (colors and texture) were conducted for the differentiation of the species within the genus Microsporum and Trichophyton. In addition, real-time PCR was applied for direct analysis of the fungal DNA obtained from the hair sample. Microscopic examination was negative. The dermatophyte present in the hair sample was confirmed as Trichophyton mentagrophytes by culture and qPCR (melting-point analysis). The patient was treated with systemic itraconazole [10 mg/kg SID - 90 days]. Twice-weekly application of 2.5 % miconazole and 2% chlorhexidine shampoo until complete cure.Discussion: Dermatophytosis is often listed as self-limiting infection; however, animal dermatophytosis can spread between pets, as well as a zoonotic transmission to humans. The literature on dermatophytosis indicates that Microsporum canis is the predominant etiological agent, followed by M. gypseum. Trichophyon mentagrophytes that appear in a lower percentage of isolation. The culture of hair, even with specific medium containing chloramphenicol and cyclohexamide, may present contaminating fungi, not related to dermatophytosis, which can inhibit or override the growth of dermatophytes. The use of real-time PCR provided a faster and specific diagnosis of dermatophytosis when compared to the conventional mycological methodology for detection and identification of T. mentagrophytes, which takes around 10 to 15 days for culture. It is possible to use this technique as an alternative diagnosis for dermatophytes associated to clinical hair samples of dogs. Keywords: dermatophytosis, dog, pets, qPCR, hair samples, diagnosis, molecular methodology

Bovine ringworm : detection of Trichophyton verrucosum by SYBR-Green real-time PCR

Trichophyton verrucosum is the most commonly dermatophyte involved in cattle ringworm. This work reported a case of bovine dermatophytosis due to Trichophyton verrucosum detected from the clinical sample by SYBR-Green real-time PCR. The strategy was based on the DNA extraction directly from the infected hair followed by realtime PCR and melting-point analysis. A faster and differential diagnosis was observed when compared to the conventional mycological methodology for detection and identification of Trichophyton verrucosum

Production and evaluation of albino mutants of Sporothrix schenckii

Melanin is an important factor intrinsically related to the virulence of the Sporothrix schenckii. Mutant albino conidia are usually used as inoculants for the evaluation of the properties of melanin in experimental models for the study of sporotrichosis. The goals of this study were to analyze the presence of melanin in S. schenckii conidia, and to produce and evaluate mutant albino forms of S. Schenckii conidia. Four distinct isolates were cultured in PDA, supplemented or not with triciclazole, and in YEPD. A portion of the samples was exposed to 300 ergs/mm2 of UV radiation to generate mutants albino forms. Following the incubation, cultures were analyzed, CFU were determined, and fragments were removed for the ultrastructural analysis, to be cultured once more in YEPD e BHI. Albino colonies were exposed to visible light; all S. schenckii isolates produced melanin, and survival and viability were 66% and 96% for albino conidia. Melanin granules were visible in the cell wall of pigmented conidia, but not in albino conidia. All conidia reverted to both the pigmented and the yeast forms, and albino colonies photoreactivated. All isolates analyzed in this study generated DHN melanin. Moreover, despite the exposure to UV radiation (300 ergs/mm2) being lethal to a significant portion of the colonies, the viability and conversions to the pigmented and yeast forms in surviving colonies did not appear to be affected. Also, albino colonies photoreactivated upon exposure to visible light

Comparison of different culture media for mycological evaluation of commercial pet food

The choice of a culture media to proceed a mycological analysis of food is extremely important to guarantee the reliability of the analysis. The medium should allow an excellent recovery of fungal species present in the food at the same time that it is necessary to avoid bacterial development. Thus the real microbial ecology will be reflected in the results. The efficacy of three media for fungal isolation and quantification (potato dextrose agar, dichloran rose of bengal and chloramphenicol agar and dichloran glycerol 18% agar) was compared for analysis of 54 samples of dry commercial pet food (34 for dogs and 20 for cats). Fungi were present in 74% of samples and 23 genera were isolated and identified. Aspergillus sp. and Aspergillus niger were respectively the most frequent genera and species isolated, whatever the culture medium selected to proceed the analysis. Dichloran glycerol 18% agar was the medium that presented the best results considering both the quantity and variety of isolated fungi. Comparing the results obtained in different media, it was observed that the fungi  recovered can vary according to the selected culture medium. Eurotium was the genus that presented the biggest difference in occurrence among the media where samples were cultivated to enumeration in this study. Therefore, the utilization of specific media, selected according to the characteristics of the food to be analyzed (specially water activity) allow a better visualization of the presence of food spoilage fungi

Imunidade humoral em caninos e felinos com dermatofitose

38

Pesquisa de criptococose em cães atendidos no Hospital de Clínicas Veterinárias da UFRGS, Porto Alegre, Brasil

A criptococose, micose causada por espécies encapsuladas do gênero Cryptococcus, acomete o homem e vários animais, podendo ocorrer em indivíduos imunocompetentes, mas freqüentemente está associada a um estado de comprometimento imunológico. A via mais freqüente de contaminação por esta levedura é a inalatória. Foi pesquisada a ocorrência do Cryptococcus em cães com sintomatologia respiratória e/ou neurológica, atendidos no Hospital de Clínicas Veterinárias da Universidade Federal do Rio Grande do Sul (UFRGS). A amostragem foi composta por 112 cães. O exame direto foi realizado em apenas 17 amostras de líquido cefalorraquidiano (LCR), e o cultivo em ágar níger (Guizotia abissynica) a 37°C durante dez dias, a partir de secreção nasal (112), sangue total (112) e LCR (n=17). Para a aglutinação em látex (LA), utilizou-se o kit Crypto-LA (Wampole), para testar soro (n=112) e LCR (n=17). O teste também foi realizado com tratamento prévio com pronase em 36 amostras (32 soros e 4 LCR). Os resultados dos exames direto, cultivo e LA foram negativos para o Cryptococcus em todas as amostras testadas. Apesar da negatividade dos resultados desta amostragem, é plausível a expectativa da ocorrência da infecção pelo Cryptococcus posto que, existe uma população canina cons tantemente exposta ao risco de se contaminarem com propágulos fúngicos, presentes nos excrementos de pombos que coabitam, em grande número, na região de abrangência do estudo

Canine ringworm caused by Trichophyton mentagrophytes : detection by SYBR-Green real-time PCR

Background: Dermatophytes, fungi of universal distribution, invade semi or fully keratinized structures, such as skin, fur/ hair and nails. The various species of dermatophytes are classified into three genera anamorphic: Microsporum, Trichophyton and Epidermophyton. The genus Epidermophyton includes only E. floccosum, that rarely affects animals. The main species responsible for the disease in dogs and cats are Microsporum canis, M. gypseum and Trichophyton mentagrophytes, which were characterized through conventional mycological methodology (microscopic examination with KOH and culture). Molecular methodologies, such as real-time PCR, can contribute to a rapid laboratory diagnosis, helping clinicians to initiate an early antifungal treatment. This case report describes a case of canine dermatophytosis due to Trichophyton mentagrophytes detected from a clinical sample by SYBR-Green real-time PCR. Case: A 8-year-old dog, rescued from the street, was referred to a private veterinary clinic in the city of Canoas, RS, Brazil, presenting generalized lymphadenomegaly, crusted lesions all over the body, generalized alopecia, signs of excoriation and epistaxis. Initially, were administered prednisone [1 mg/kg every 48 h, BID] and cephalexin [30 mg/kg, BID]. Weekly baths with benzoyl peroxide were also given. The therapy was not clinically successful. Wood’s Lamp Test was negative. As a differential diagnosis, PCR for detection of Leishmania was negative. Complete blood count and serum biochemical assay were also performed. For mycological diagnosis, hair specimen was clarified and examined microscopically using 10% potassium hydroxide (KOH) for the visualization of chains of arthroconidia (ectothrix invasion of hair). The infected hair was plated onto MycoselTM Agar, incubated at 28°C for 15 days. Microscopy of hyphae/ conidia and macroscopic colony characteristics (colors and texture) were conducted for the differentiation of the species within the genus Microsporum and Trichophyton. In addition, real-time PCR was applied for direct analysis of the fungal DNA obtained from the hair sample. Microscopic examination was negative. The dermatophyte present in the hair sample was confirmed as Trichophyton mentagrophytes by culture and qPCR (melting-point analysis). The patient was treated with systemic itraconazole [10 mg/ kg SID - 90 days]. Twice-weekly application of 2.5 % miconazole and 2% chlorhexidine shampoo until complete cure. Discussion: Dermatophytosis is often listed as self-limiting infection; however, animal dermatophytosis can spread between pets, as well as a zoonotic transmission to humans. The literature on dermatophytosis indicates that Microsporum canis is the predominant etiological agent, followed by M. gypseum. Trichophyon mentagrophytes that appear in a lower percentage of isolation. The culture of hair, even with specific medium containing chloramphenicol and cyclohexamide, may present contaminating fungi, not related to dermatophytosis, which can inhibit or override the growth of dermatophytes. The use of real-time PCR provided a faster and specific diagnosis of dermatophytosis when compared to the conventional mycological methodology for detection and identification of T. mentagrophytes, which takes around 10 to 15 days for culture. It is possible to use this technique as an alternative diagnosis for dermatophytes associated to clinical hair samples of dogs

Quérion causado por Microsporum gypseum em um cão

Quérion (dermatofitose nodular) é uma apresentação clínica de micose cutânea caracterizada por uma dermatite profunda que varia de uma reação granulomatosa até piogranulomatosa, com aspecto de nódulo edematoso, circular e alopécico. Em animais é causado principalmente pelas espécies de dermatófitos Microsporum gypseum, M. canis e Trichophyton mentagrophytes. Microsporum gypseum é um fungo geofílico, cosmopolita e sua transmissão ocorre por contato com solos contaminados. É relatado um caso de lesão do tipo quérion causada por M. gypseum em um cão da raça Dachshund, fêmea, 4 anos de idade, atendido no Hospital de Clínicas Veterinárias da Universidade Federal do Rio Grande do Sul, com uma lesão circular na região do focinho medindo 4 cm de diâmetro. Por se tratar de uma lesão dermatofítica com infecção bacteriana e muito prurido, optou-se pelo tratamento tópico com creme contendo associação medicamentosa de miconazol, gentamicina e betametasona, duas vezes ao dia, por quarenta e cinco dias, havendo regressão completa do quadro, sem recidiva clínica. É esentada uma extensiva revisão de casos similares observados em outros países. Aparentemente trata-se do primeiro caso de quérion registrado em animais na literatura veterinária brasileira com a respectiva identificação do dermatófito