A simplified method to produce Paracoccidioides brasiliensis exoantigens for immunodiffusion testing is proposed. It uses technology accessible for small laboratories with few resources in Latin America, where paracoccidioidomycosis is endemic. This procedure may replace the more complex procedure, originally proposed by Camargo et al. in 1988, that is currently commonly used. It is based on the production of exoantigen by P. brasiliensis isolate B339, a good secretor of the characteristic 43000-Da glycoprotein gp43.

We used RAPD (random amplification of polymorphic DNA) to analyze the phenotypic and genotypic characteristics of Sporothrix schenckii isolated from five geographic regions of Brazil, from clinical, animal, and environmental sources. Our results yielded a significant difference (P<0.01) in the mean conidial area of S. schenckii animal isolates (2.96 ± 1.07) compared with those of clinical isolates (fixed form, 2.33 ± 0.53; lymphocutaneous form, 2.37 ± 0.43). There was no association among S. schenckii clinical isolates and geographic region. Isolates from the Northeast region exhibited the lowest thermotolerance (% growth inhibition) at 35ºC (x = 49.23% ± 17.25) and at 37ºC (70.43% ± 10.93%). Northern isolates exhibited the highest thermotolerance at 35ºC (12.82% ± 5.73%) and at 37ºC (23.81% ± 8.27%). RAPD with a 10-mer primer OPD-18 generated 67 PCR fingerprint patterns. The 151 S. schenckii isolates fell into seven major clusters with such great genetic diversity that an association of isolates with clinical forms or geographic areas could not be determined, even with investigations focused on more restricted geographic areas. The main physiological characteristics of Brazilian S. schenckii isolates were also characterized, including osmophilia, halophilia, pH tolerance, urease activity, casein hydrolysis, and gelatinase, proteinase, and DNAase production.

There is some evidence that dogs can be naturally infected by Paracoccidioides brasiliensis in endemic areas of paracoccidioidomycosis. In order to evaluate canine infection with this fungus, a survey with 149 urban and 126 rural dogs was carried out using ELISA and intradermal tests with the gp43 antigen of P. brasiliensis in Uberaba, Minas Gerais state of Brazil. Forty-one out of 149 urban dogs were euthanatized and had their lungs, liver and spleen removed. One slice from each viscera was processed for histopathological examination and the remaining was homogenized and then cultivated on mycobiotic agar at room temperature and Fava-Netto medium at 35 degrees C and observed for 12 weeks. Of urban dogs, 75 (50.3%) were small adult females, 56 (36%) were strays, while 93 (64%) had been donated to the municipal zoonosis control center. Nine (6.2%) had a positive intradermal test without statistical differences regarding gender, race, nutritional status or origin. No colonies with microscopic or morphology appearances resembling P. brasiliensis were isolated, nor granulomatous process or fungal structures were observed from histopathological examination. Eighty (53.6%) of the urban dogs presented seroreactivity, without statistical differences regarding gender, race, nutritional state, origin, or positive intradermal test. Of 126 rural dogs, 102 (80.5%) presented antibodies against gp43 antigen, and this was statistically significant in relation to the reactivity detected in urban dogs (P = 0.0001). Thus, dogs are commonly infected with P. brasiliensis, but they probably present natural resistance to develop paracoccidioidomycosis.

Background Paracoccidioidomycosis (PCM) is a systemic mycosis commonly found in Latin America that is caused by distinct species of Paracoccidioides genus: Paracoccidioides brasiliensis complex (S1, PS2, PS3 and PS4) and Paracoccidioides lutzii . Its pathobiology has been recently explored by different approaches to clarify the mechanisms of host-pathogen interactions underpinning PCM. The diversity of clinical forms of this disease has been attributed to both host- and fungus-related factors. Methodology/Principal findings For better understanding of the molecular underpinnings of host-fungus interactions, we evaluated in vivo virulence of nine Paracoccidioides brasiliensis complex isolates and correlated it to protein expression profiles obtained by two-dimensional gel electrophoresis. Based on the recovery of viable fungi from mouse organs, the isolates were classified as those having low, moderate, or high virulence. Highly virulent isolates overexpressed proteins related to adhesion process and stress response, probably indicating important roles of those fungal proteins in regulating the colonization capacity, survival, and ability to escape host immune system reaction. Moreover, highly virulent isolates exhibited enhanced expression of glycolytic pathway enzymes concomitantly with repressed expression of succinyl-CoA ligase beta chain, a protein related to the tricarboxylic acid cycle. Conclusions/Significance Our findings may point to the mechanisms used by highly virulent P . brasiliensis isolates to withstand host immune reactions and to adapt to transient iron availability as strategies to survive and overcome stress conditions inside the host.

Prematurely-born infants are exposed to multiple invasive procedures while in the intensive care unit. Newborn rats and humans have similar behavioral responses to noxious stimulation. Previous studies have shown that early noxious stimuli may alter dentate gyrus neurogenesis and the behavioral repertoire of adult rats. We evaluated the late effects of noxious stimulation administered during different phases of development on two spatial memory tests; object recognition (OR) and Morris water maze (WM) tests. Noxious stimulation was induced by an intra-plantar injection of complete Freund's adjuvant (CFA) on postnatal (P) day 1 (group P1) or 8 (P8). Control animals were not stimulated. Behavioral tests were conducted on P60 in both male and female animals. In the WM, three domains were evaluated: acquisition, probe trial performance and reversal re-acquisition. The number of Nissl stained cells in the dentate granule cell layer was assessed by stereological counting. The OR test revealed that P1 male rats had poor long-term memory compared to the control and P8 groups. In the WM, no short- or long-term memory differences were detected between early postnatal-stimulated male and female rats and their respective controls. However, the ability to find the hidden platform in a new position was reduced in P1 male rats. The number of dentate granule cells in P8 males was higher than in all other groups. This study demonstrates that noxious stimulation on P1 results in spatial learning deficits in male animals, but does not disrupt the development of the hippocampus-dependent strategies of learning and memory.