XI International Meeting on Paracoccidioidomycosis

XI International Meeting on Paracoccidioidomycosis

Resume:27-1

Poster (Painel)

27-1	Chlamydospores of Paracoccidioides brasiliensis: isolation and study of infectivity in a murine experimental model
Authors:	Igor Otavio Minatel (UNESP - Universidade Estadual Paulista - Júlio de Mesquita Filho) ; Denise Fecchio (UNESP - Universidade Estadual Paulista - Júlio de Mesquita Filho) ; Julio Defaveri (UNESP - Universidade Estadual Paulista - Júlio de Mesquita Filho)

Abstract

Introduction: Paracoccidioides brasiliensis (Pb) is the causative agent of Paracoccidioidomycosis (PCM), a systemic mycosis endemic in Latin America. The mode of infection is not known, but it is accepted that it occurs after inhalation of asexual propagules produced in an adverse environmental conditions such as nutrient deprivation and variations in temperature. Pb growing at room temperature (~25°C) produces few chlamydospores and when the temperature is increased up to 32°C, great number of chlamydospores is produced. In endemic areas of PCM are common temperature variations, thus creating environmental conditions to production of chlamydospores. In the absence of studies addressing the isolation and the potential infectivity of chlamydospores, this work aimed to develop a protocol for production, isolation and purification of chlamydospores and study their potential infectivity in mice. Methodology: The Bt83 strain was grown on potato broth for 14 days at 25°C and later incubated for 36h at 32°C. The chlamydospores were released from mycelium by magnetic stirring (400 rpm) and sonication (22.5 kHz for 5 seconds) and then purified by 95% Percoll gradient centrifugation. Viability was assessed by fluorescein technique. To assess the potential infectivity of chlamydospores, 8 weeks old male swiss mice, were instilled intranasally with 106 chlamydospores and euthanized after 24h, and 1, 2 and 4 weeks of infection. Lungs, spleen and liver were removed for histopathological analysis and recovery of colony forming units. Results: Incubation at 32°C stimulated the large formation of chlamydospores, which maintained intact their morphological features and preserved viability of 90% at the end of purification. Twenty-four hours after infection the lungs showed neutrophilic bronchopneumonia foci, containing large numbers of yeasts. In subsequent periods there were many small epithelioid granulomas, with the presence of fungi and lymphocytes. Dissemination to the spleen (100%) and liver (83%) was an early event, observed from 1st week of infection. Conclusion: The processes of production, isolation and purification resulted in suspension ideal for experimental study. The results show that the chlamydospores are highly infectious, capable of inducing early dissemination and can play an important role in infection and development of paracoccidioidomycosis.

Keyword: Chlamydospore, Infection, Isolation, Paracoccidioides brasiliensis, Purification

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