Leveduras isoladas do pelame de cães sadios que vivem em regime domiciliar / Yeasts isolated from the haircoat of healthy dogs that live indoor

Potentially pathogenic yeasts from the haircoat of dogs that live indoor and visit regularly an esthetic service were collected by the carpet technique applied on the fur. The microorganisms were isolated on Sabouraud dextrose agar supplied with chloramphenicol and identified by their morphological and biochemistry characteristics. Candida albicans was identificated in 95.2% of the canine population, followed by Rhodotorula mucilaginosa (4.8%). It is known that C. albicans is uncommon on health skin. By the other hand, the high frequency of this agent in this study may represent exposure and risk for opportunistic infections in dogs and other susceptible animals.

Allergenic extracts from Metarhizium canisopliae: obtainment and characterization.

Metarhizium anisopliae is used as a biopesticide for insects that damage agricultural plantations like sugar cane and forage plants. In a previous study the sensitization to this fungus of asthmatic patients coming from sugar cane areas was showed. The aims of this work were: to compare crude extracts obtained with Tris-HCl and Coca liquid from several growth phases of M. anisopliae concerning the total content of proteins and their electrophoretic analysis profile; to evaluate in vivo allergic sensitization in Balb/c mice and allergic patients from a sugar cane area, and to characterize the allergenic fractions in the sera of patients positive for the prick test by means of Western-blotting. The extract obtained with Coca liquid on the 16th day was the one that presented the greatest number of proteic fractions, including all those present in the other extracts. Twelve fractions were verified in this extract with approximate molecular weights from 94 to 14 kDa. The allergenicity of the extract obtained on the 16th day was proven by the production of IgE antibodies in Balb/c mice, with titres of 200. Prick tests carried out with the extract of the 16th day in 79 atopic individuals (from sugar cane area), 35 atopic individuals (from urban area) and 11 non- atopic individuals showed respective positivity of 29%, 9% and 0%. The allergenic characterization in vitro was performed by means of Western blotting, and the fractions that reacted with the positive individuals' sera were those of approximate molecular weights of 67 kDa (95%); 20 kDa (55%); 94 kDa (36%); 34 and 36 kDa (23%); 43 and 48 kDa (14%); 16 kDa (9%) and 54kDa (5%). It was concluded that the crude allergenic extract, obtained with Coca liquid from the 16th day growth of Metarhizium anisopliae, contains allergenic fractions and can be used in diagnostic screening tests.

Trichophyton rubrum isolated from aids and human immunodeficiency virus-infected patients in São Paulo, Brazil: antifungal susceptibility and extracellular enzyme production.

BACKGROUND: In order to identify intraspecific variations in Trichophyton rubrum and to correlate them to the immunological status of the host, sixty strains isolated from AIDS, HIV-positive and HIV-negative patients were compared for the production of extracellular enzymes and for their susceptibility to several antifungal drugs. METHODS: The isolates were tested for their ability to secrete keratinases, proteinases, phospholipases, lipases and DNases. Likewise, we investigated their susceptibility to amphotericin B, ketoconazole, ciclopiroxolamine, griseofulvin, miconazole and tolnaftate. RESULTS: Variations in the Minimal Inhibitory Concentration (MIC80)) values were observed for all antifungals tested, but they were similarly distributed among the three clinical groups. Griseofulvin showed the most prominent differences among the three groups of isolates. Regarding enzyme secretion, all samples secreted keratinases and DNases, while none secreted phospholipases. Proteinases and lipases were secreted by some of them. CONCLUSIONS: The differences among isolates of the three groups were not statistically significant and therefore could not be ascribed to a given clinical status. Intraspecific variations similarly occurred in each group, irrespective of the immunological status of the patients.

Criptococose canina: relato de caso / Canine ocular cryptococcosis: a case report

Descreve-se, pela primeira vez no Brasil, um caso de criptococose canina com acometimento oftalmo e dermatopático, com diagnóstico intra-vitam, em animal da raça Pastor Alemão, fêmea, com 24 meses de vida, criado em São Paulo, em contacto com outros cinco cães assintomáticos, que havia se infectado pelo contato com dejetos de pombos (Columba livia). Evoluia há 90 dias, com quadro tegumentar e ósseo, sintomas e lesões características, sendo, ainda, detectada forma assintomática de coriorretinite, de início unilateral. O diagnóstico foi estabelecido pelos dados da anamnese, dos exames físico, dermatológico e complementares (radiográfico, cultivo micológico, histopatologia de pele) tendo-se evidenciado e isolado cepa de Criptococcus neoformans var. neoformans. Após nove meses de terapia com itraconazol (9mg/kg/SID/VO) houve involução total do quadro, sem qualquer efeito adverso à droga.

Biosynthesis of chondroitinase and hyaluronidase by different strains of Paracoccidioides brasiliensis.

The biosynthesis of chondroitinase and hyaluronidase by different isolates of Paracoccidioides brasiliensis was investigated in 20 strains isolated from patients (17 strains), a penguin (Pygocelis adeliae, one strain), an armadillo (Dasypus novemcinctus, one strain) and the environment (dog food, one strain). All the P. brasiliensis isolates studied had the ability to produce chondroitinase and hyaluronidase, although differences in colony morphology and enzyme production were detected among them. These results suggest that further investigations should be carried out in the clinical field in order to clarify the potential role of P. brasiliensis enzyme production in the pathogenesis of paracoccidioidomycosis.

Comparative performance of two air samplers for monitoring airborne fungal propagules

Many studies have attempted to evaluate the importance of airborne fungi in the development of invasive fungal infection, especially for immunocompromised hosts. Several kinds of instruments are available to quantitate fungal propagule levels in air. We compared the performance of the most frequently used air sampler, the Andersen sampler with six stages, with a portable one, the Reuter centrifugal sampler (RCS). A total of 84 samples were analyzed, 42 with each sampler. Twenty-eight different fungal genera were identified in samples analyzed with the Andersen instrument. In samples obtained with the RCS only seven different fungal genera were identified. The three most frequently isolated genera in samples analyzed with both devices were Penicillium, Aspergillus and Cladophialophora. In areas supplied with a high efficiency particulate air filter, fungal spore levels were usually lower when compared to areas without these filters. There was a significant correlation between total fungal propagule measurements taken with both devices on each sampling occasion (Pearson coefficient = 0.50). However, the Andersen device recovered a broader spectrum of fungi. We conclude that the RCS can be used for quantitative estimates of airborne microbiological concentrations. For qualitative studies, however, this device cannot be recommended

Comparative performance of two air samplers for monitoring airborne fungal propagules.

Many studies have attempted to evaluate the importance of airborne fungi in the development of invasive fungal infection, especially for immunocompromised hosts. Several kinds of instruments are available to quantitate fungal propagule levels in air. We compared the performance of the most frequently used air sampler, the Andersen sampler with six stages, with a portable one, the Reuter centrifugal sampler (RCS). A total of 84 samples were analyzed, 42 with each sampler. Twenty-eight different fungal genera were identified in samples analyzed with the Andersen instrument. In samples obtained with the RCS only seven different fungal genera were identified. The three most frequently isolated genera in samples analyzed with both devices were Penicillium, Aspergillus and Cladophialophora. In areas supplied with a high efficiency particulate air filter, fungal spore levels were usually lower when compared to areas without these filters. There was a significant correlation between total fungal propagule measurements taken with both devices on each sampling occasion (Pearson coefficient = 0.50). However, the Andersen device recovered a broader spectrum of fungi. We conclude that the RCS can be used for quantitative estimates of airborne microbiological concentrations. For qualitative studies, however, this device cannot be recommended.

Yeasts isolated from blood and catheter in children from a public hospital of São Paulo, Brazil.

The great majority of nosocomial fungal infections, especially fungemias, are caused by yeasts, mostly of the genus Candida. In addition, such infections may be associated with intravascular catheters. In this study, 80 yeast strains were isolated from hospitalized children, being 59 from blood cultures and 21 from vascular catheter cultures. The prevalent species in both blood and catheter was C. parapsilosis (32.2% and 48.9%, respectively), followed by C. albicans (16.9% and 28.6%, respectively). Concerning enzyme production, 78.8% of the 80 isolates presented strong proteolytic activity but 78.8% showed no phospholipase activity. We also detected two prevalent "killer" biotypes: 511 and 888. Additionally, in five patients, it was possible to observe that the yeast species, "killer" biotype and proteolytic and phospholipase activity of blood and catheter were similar. In view of this, we suggest a transmission of nosocomial yeast infection from catheter to blood.

Allergenic evaluation of Malassezia furfur crude extracts.

Crude extracts of the lipophilic yeast Malassezia furfur were obtained from 2, 6, 10 and 28 day old cultures. The in vitro cultivation periods corresponded, respectively, to the lag phase, middle of the log phase, end of log phase and the decline phase of the growth curve, which was based on viable cell counts obtained with a fluorescent viability test. Biochemical analyses showed that the protein and carbohydrate contents were greater in day 10 extracts. Seventy patients with different allergic manifestations and 30 healthy volunteers were skin prick tested using the extracts. Of these, thirteen (18.57%) patients gave positive responses. SDS PAGE gradient electrophoretic profiles of the preparations indicated that the 28 day extracts contained the greatest number of protein bands with molecular weights ranging mostly between 30 and 94 kDa. Immunoblots incubated with individual patient sera showed that four IgE binding M. furfur allergens of approximately 88, 61, 52 and 39 kDa were present in the 28 day extracts. The components identified could be used for detecting IgE mediated responses to M. furfur among individuals affected with different allergic conditions.

Sensitization to the fungus Hemileia vastatrix (coffee leaf rust).

BACKGROUND: The aim of the present study was to determine the existence of sensitization to the fungus Hemileia vastatrix (coffee leaf rust) in a sample of the Brazilian population. This fungus attacks coffee plantations, and in some regions of Brazil the concentration of its spores in the air can be very high. METHODS: A total of 378 individuals underwent skin tests (prick and intradermal tests) with H. vastatrix extract. The subjects were divided into four groups according to the occurrence of atopy and the region where they lived (coffee-growing or non-coffee-growing regions), and another group (V) consisted of 50 rural workers employed on coffee plantations. The presence of specific IgE against the fungus H. vastatrix in sensitized individuals was demonstrated in vitro by immunoblotting. RESULTS: There was no statistical difference in comparing the results with intradermal or prick tests. The incidence of positive tests was significantly higher among atopic individuals residing in coffee-growing regions (14.7%). Among rural workers, 10% showed positive tests. Immunoblotting revealed specific IgE against 20 protein bands of H. vastatrix. CONCLUSION: The fungus H. vastatrix may be an important allergen in coffee-producing countries. The sensitization is more frequent among atopic individuals residing in coffee-growing regions.

Phenotypic characterization of Trichophyton rubrum isolates from two geographic locations in Brazil.

To characterize possible Trichophyton rubrum phenotypes, which circulate in two Brazilian localities, we tested 53 isolates of this dermatophyte for their ability to assimilate several carbon sources, for keratinase, proteinase, phospholipase, lipase and desoxiribonuclease (DNase) secretions, and for their susceptibility to the antifungals fluconazole, ketoconazole and itraconazole. For each method, the isolates were submitted to similarity analysis and the methods were evaluated for their discriminatory indexes. None of the isolates were capable of assimilating arabinose, dulcitol, lactose, melibiose, ribose and xylose, while all of the isolates assimilated maltose, sucrose and sorbitol. However, adonitol, cellobiose, dextrin, erythritol, fructose, galactose, inulin, mannitol, mannose, raffinose, rhamnose and trehalose were assimilated by some isolates but not by others. All isolates secreted keratinase and DNase, while none secreted phospholipase. Proteinase and lipase were secreted only by some isolates. All but four isolates were resistant to fluconazole, most of them were sensitive to ketoconazole and all were sensitive to itraconazole. Carbohydrate assimilation was the method that presented the highest discriminatory index, and also the method that displayed the largest number of biotypes. Taken together, these data suggest that significant phenotypic variations exist among T. rubrum isolates. They seem to occur independently from their geographic origins.

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.

Production of extracellular enzymes by Microsporum canis and their role in its virulence.

Microsporum canis is the most prevalent dermatophyte of domestic animals. Several enzymes produced by dermatophytes, particularly keratinases, are considered to play a role in the virulence of this fungus. To investigate the possible relationship between the clinical status of M. canis infection and enzymatic activity of isolates, we studied the relationship between keratinase, elastase, lipase and DNase levels produced in vitro by different isolates and virulence as expressed in a guinea pig model. Samples isolated from symptomatic dogs and cats showed a statistically significantly (P < 0.05) higher keratinase activity than samples isolated from asymptomatic animals. Experimental infection of guinea pigs showed that a strain with high in vitro keratinase activity induced acute infection, which resolved clinically and mycologically faster than the infection induced by a strain with low keratinase activity. This suggested a strong correlation between high keratinase activity and the development of symptoms. The same correlation was not observed for other enzymes tested.

Production of proteinase and phospholipase by Paracoccidioides brasiliensis.

We have investigated the production of proteinase and phospholipase by 20 different isolates of Paracoccidioides brasiliensis. Isolates were grown in Bacto-peptone, Dextrose, pH 5.5, agar slants, at 27 degrees C for 30 days, and cultures were transferred onto Petri dishes containing basis medium and bovine serum albumin fraction V and sterile egg yolk as substrates for enzyme production, and incubated at 27 degrees C. After 30 days net enzyme activity was visualized and quantitatively evaluated, measuring a ratio between colony diameter and diameter of the transparent (proteinase) or white (phospholipase) ring zone surrounding it. Results demonstrated that all isolates had the ability to produce proteinase and phospholipase, even though variability in enzyme production was noted among different isolates of P. brasiliensis.

Characterization of allergenic fractions from Drechslera monoceras.

Drechslera monoceras, a fungus of the Deuteromycota phylum, is fairly frequent in Brazil, and is spread through the atmosphere. In previous studies done in the city of Sao Paulo, it was found that in relation to 42 other fungi extracts, the crude extract of this fungi demonstrated a more intense cutaneous reaction in patients with respiratory allergies. Biochemical, antigenic and allergenic evaluations were carried out at various growth stages of this fungus. Based on these facts, the purpose of this research was the fractionation and allergenic characterization of the allergenic extract of D. monoceras to be used in diagnosis and immunotherapy in patients with positive cutaneous reaction to this fungus. In the city of Sao Paulo, 13 of 248 patients with respiratory allergy (asthma and/or rhinitis) showed positive reaction following cutaneous tests (skin prick tests). The crude extract of D. monoceras was fractionated by SDS-PAGE. The visible fractions were then separated by electroelution to be inoculated into BALB/c mice to evaluate the production of IgE antibody. The IgE content was detected by passive cutaneous anaphylaxis test in Wistar rats, and two fractions of approximate molecular weights of 14.4 and 36 KDa reacted to the test. The in vitro allergenic characterization was carried out by Western blotting, and three fractions of approximate molecular weights of 14.4, 36 and 60 KDa were positive. It was concluded that the extract of D. monoceras has at least three allergenic determinants, which can be used for diagnosis and immunotherapy in patients with respiratory allergy to this fungi.

Evaluation of the allergenicity of spore and mycelia extracts of Pisolithus tinctorius.

The antigenic and allergenic chemical analysis of spore and mycelia extracts of Pisolithus tinctorius was carried out. The spores were collected from basidiocarps in plantations of Eucalyptus spp and the mycelia from culture in MNM medium. With basis on the fungus growth curve, the mycelia masses were obtained after 10, 20, 30, and 40 days of incubation, which correspond, respectively, to the beginning, middle and end of the log phase, and beginning of the decline phase. The mycelia masses, together with the spores, were submitted to the action of three extractors (Coca, Tris-HCl, and ammonium bicarbonate). The contents of carbohydrates and proteins were determined. The SDS-PAGE electrophoretical analysis revealed separate fractions in these extracts, besides common fractions, in function of cultivation time and extraction methods. The selected extracts for the allergic tests were the ones with the highest number of fractions. The prick-tests were conducted in 374 patients--rural workers, eucalyptus plantation workers, and college students. The positivity to the "prick test" with the antigenic extract of P. tinctorius was, respectively, 3.78%, 28.20% and 6.40%. Most prick-test positive patients (82.75%) also presented symptoms of respiratory allergy (asthma and rhinitis). There was no reactivity difference when the spore and mycelia extracts were employed. The analysis of the positive patients' sera revealed the presence of IgE specific to the P. tinctorius antigens. Since Pisolithus tinctorius is found as mycorrhiza of Eucalyptus spp, and this plant is used in reforestation in most countries, the importance of that fungus should be regarded as a possible cause of respiratory allergies, especially in occupationally exposed workers.

Candidin in immediate hypersensitivity. Comparison of two antigens.

One hundred outpatients in a Clinic of Allergy were submitted to intradermic tests with two types of candidins. The main focus of the research was the comparison of two antigens obtained from the same strains of Candida albicans: one a suspension of yeast cells and the other, a polysaccharide. The readings, taken 20 minutes after the intradermic injections, with positive results were considered as hypersensitivity of the immediate type. Positive results were obtained in 74% of the patients with the yeast cell antigen and in 73% with the polysaccharide antigen. This research mainly deals with the advantages that can be obtained by using the polysaccharide antigen in intradermic tests for evaluating hypersensitivity of the immediate type.

Candidin: comparison of two antigens for cutaneous delayed hypersensitivity testing.

A candidin, which is a suspension of killed yeast cells, is commonly used for intradermal tests of delayed hypersensitivity, to evaluate the immunological cellular competence of the patient, when the test is applied along with other similar tests. When working with a cellular antigen, the histopathology of positive skin tests reveals a cellular infiltrate which not only presents a characteristic hypersensitivity reaction but also a neutrophilic abscess in the central part. This research presents the results of a comparison between the yeast cell suspension and the polysaccharide antigens, both obtained from the same strains of Candida albicans. The results obtained by skin tests in one hundred individuals were 61.0% with the polysaccharide antigen and 69.0% with the yeast cell suspension antigen. Concordant results concerning the two antigens were observed in 82.0% of the individuals. The discussion section presents an assumption to explain the differences of positivity obtained with the two antigens. We conclude that the polysaccharide antigen can be utilized in the intradermal test of delayed hypersensitivity to Candida albicans.

Acute effect of aflatoxin B1 on different inbred mouse strains II.

The acute effects of Aflatoxin B1(AFB1) were evaluated on C57B1/6, CBA/J, and Balb/c mice challenged with a single intraperitoneal dose of the mycotoxin (60 mg/Kg animal weight). 90 mice per strain were divided into three groups of 30 animals each: the intoxicated group and control groups I and II. Intoxicated mice were injected intraperitoneally with AFB1 dissolved in corn oil, while control I mice received corn oil only (0.01 ml/g) by the same route. Lots of 10 animals from the intoxicated and control groups were sacrificed 24, 72 and 168 hours after challenge. Control mice II remained untreated and were used as standards of normality for biochemical (hepatic and renal function) and hematological evaluation. AFB1 was detected in the liver of C57B1/6 and CBA/J mice 24 hours (1.46 and 0.75 ng/g, respectively), 72 hours (2.30 and 0.08 ng/g, respectively), and 168 hours (2.18 and 0.25 ng/g, respectively) after challenge. The mycotoxin was also observed in the liver of B10A mice (6.20 ng/g) 72 hours post-injection. The most evident histological lesions were observed 168 hours after treatment in C57B1/6 and B10A mice. Serum levels of alkaline phosphatase in intoxicated C57B1/6 and B10A mice were significantly higher than those of control I and II animals. The histopathologic lesions and biochemical changes were very discrete in Balb/c and CBA/J mice. It is included that strains C57B1/6 and B10A are more susceptible than strains CBA/J and Balb/c to the acute effects of AFB1. Such difference probably reflects each strain's ability to biotransform and eliminate AFB1 and its metabolites.