Growth-promoting factors for yeast cells ofParacoccidioides brasiliensis

Low-density seedings of yeast cells ofParacoccidioides brasiliensis give poor growth (as assessed by plating efficiency test) on conventional mycological agar media, and therefore growth-promoting factors for this fungus were sought. Water-extracts of yeast cells of sixP. brasiliensis isolates were all considerably effective in promoting the growth of low-density seedings ofP. brasiliensis isolates Pb-18 and Hachisuga, but had little effect on isolate Bt-4. Horse serum, at a concentration range of 2–4%, moderately or considerably promoted the growth of theseP. brasiliensis isolates. Combinations of the fungus cell extracts with horse serum were highly effective in promoting the growth of all of the fungal isolates. The fungus cell extracts showed siderophore (microbial iron carrier) activity. An iron-chelator, ethylenediaminetetraacetic acid, at a concentration of 100 μM also highly promoted the growth of the fungal isolates in the presence of horse serum, and ferric ion added to culture medium was considerably effective in the growth promotion. These results suggest that deficient utilization of external iron by the fungus cell is one of the growth-limiting processes for low-density seedings of yeast cells ofP. brasiliensis on conventional mycological agar media.     

Virulence of Paracoccidioides brasiliensis: The influence of in vitro passage and storage

Stability of virulence in P. brasiliensis isolates was studied with respect to the in vitro culture history and methods used for storage. Virulence in yeast-form P. brasiliensis isolates was tested in a chronic pulmonary murine model of paracoccidioidomycosis where progression of disease was quantitated in terms of colony forming units recoverable from lungs. Four isolates of P. brasiliensis, including recently isolated from patients or experimental animals, caused chronic progressive disease. Two isolates with a history of subculturing showed attenuation by causing resolving but chronic disease. An attenuated isolate became avirulent subsequent to 15 more years of subculturing. These findings suggest that virulence of P. brasiliensis can be attenuated or lost subsequent to cycles of subculturing over long periods. Our data suggest that the use of fresh P. brasiliensis isolates may be needed to provide reproducible virulence for experimental systems.Abbreviations ATCC American Type Culture Collection - CFU colony-forming units - LD Lethal dose - MMv-M modified McVeigh Morton - PMN polymorphonuclear neutrophil     

Delayed-type hypersensitivity response in an isogenic murine model of paracoccidioidomycosis

The specific delayed-type hypersensitivity (DTH) response was evaluated in resistant (A/SN) and susceptible (B10.A) mice intraperitoneally infected with yeasts from a virulent (Pb18) or from a non-virulent (Pb265)Paracoccidioides brasiliensis isolates. Both strains of mice were footpad challenged with homologous antigens. Pb18 infected A/SN mice developed an evident and persistent DTH response late in the course of the disease (90th day on) whereas B10.A animals mounted a discrete and ephemeral DTH response at the 14th day post-infection. A/SN mice infected with Pb265 developed cellular immune responses whereas B10.A mice were almost always anergic. Histological analysis of the footpads of infected mice at 48 hours after challenge showed a mixed infiltrate consisting of predominantly mononuclear cells. Previous infection of resistant and susceptible mice with Pb18 did not alter their DTH responses against heterologous unrelated antigens (sheep red blood cells and dinitrofluorobenzene) indicating that the observed cellular anergy was antigen-specific. When fungal related antigens (candidin and histoplasmin) were tested in resistant mice, absence of cross-reactivity was noted. Thus, specific DTH responses againstP. brasiliensis depend on both the host's genetically determined resistance and the virulence of the fungal isolate.Abbreviations DTH delayed-type hypersensitivity - DNFB dinitrofluorobenzene - FN18 Fava Netto's antigen obtained from isolate Pb18 - FN265 Fava Netto's antigen obtained from isolate Pb265 - SRBC sheep red blood cells     

Paracoccidioides brasiliensis antigen batches from the same isolate show immunological and biochemical differences

We investigated the occurrence of antigenic and biochemical variability among Paracoccidioides brasiliensis antigen batches prepared according to the same protocol. Initially (experiment #1), we analyzed two antigen lots of two human isolates (Bt1 & Bt2), cultured in two media (PYG: bactopeptone, yeast extract, glucose; MMM: McVeigh & Morton medium) in SDS-PAGE and in two immunological tests (imunodiffusion-ID and footpad swelling test-FPT). Afterwards (experiment #2), we compared the antigenic profile of three antigen batches from three human isolates (Bt1, Bt2 & Bt3) by two-dimensional immunoelectrophoresis (2 D-IEP) against a reference system for P. brasiliensis antigens. In experiment #1, there were important intra- and inter-strain antigenic differences between batches of the fungal isolates cultured on both media. The block titration of the antigen batches for the immunological tests revealed correlation between protein concentration and biological activity in ID and no correlation in FPT. In experiment #2, the reference system for P. brasiliensis showed 26 antigen peaks. There were important differences between batches prepared from the same isolate and between batches from different isolates. Our data suggested the occurrence of instability in the synthesis of antigenic components by a same P. brasiliensis isolate, under controlled incubation conditions.     

Studies on the relationship between the pathogenicity of Paracoccidioides brasiliensis in mice and its growth rate under different oxygen atmospheres

We performed comparative studies of the pathogenicity of six strains of Paracoccidioides brasiliensis (Bt-9, Bt-4, Pb-9, Pb-18, Bt-7 and B-1183) for young adult male ddY mice and the growth rate of each strain under different oxygen atmospheres (aerobic, micro-aerobic and anaerobic atmospheres) at 37 °C. 10⁶ units of yeast cells were intravenously injected into each mouse. The pathogenicity of each isolate was determined by a scoring system based on organ culture and histopathological findings. The growth rates under different oxygen atmospheres were determined by a scoring system in which 300 fungal units per strain were counted. The strain Bt-9 showed the greatest pathogenicity, followed by Bt-4. Pb-9 and Pb-18 had on intermediate rank of pathogenicity. Bt-7 and B-1183 were the least pathogenic of the strains tested. Except for strain Bt-7 all strains showed an excellent growth under an aerobic atmosphere. Bt-4 and Bt-9 also showed excellent growth under a micro-aerobic atmosphere, followed by Pb-9, whereas the growth of Pb-18, Bt-7 and B-1183 was limited. There was a correlation between the growth rate under a micro-aerobic atmosphere and the pathogenicity of a strain. The growth rate of P. brasiliensis under a micro-aerobic atmosphere strongly correlated to its pathogenicity.     

Diversity in Paracoccidioides brasiliensis . The PbGP43 gene as a genetic marker

Paracoccidioides brasiliensis is a temperature-dependent dimorphic fungus and the agent of paracoccidioidomycosis (PCM), which is prevalent in rural workers of Latin American countries. Until a decade ago, most of the studies involving P. brasiliensis used clinical isolates, since environmental samples from soil are difficult to obtain. More recently, P. brasiliensis has been isolated from infected wild and domestic animals, especially from the nine-banded armadillo Dasypus novemcinctus in Brazil. Over the years, diversity within the species has been observed at several phenotypic levels. The present review will discuss the reports focusing on genetic polymorphism, which culminated with the detection of P. brasiliensis phylogenetic species as a result of a multilocus study. Polymorphism in the PbGP43 gene is detailed. This gene encodes fungal glycoprotein gp43, a dominant P. brasiliensis antigen largely studied in the last two decades for its importance in diagnosis, immune protection, and adhesive properties to extracellular matrix-associated proteins. Fungal traits associated with genetic groups are discussed.     

Scaling up tests on virulence of the cassava green mite fungal pathogen <Emphasis Type="Italic">Neozygites tanajoae</Emphasis> (Entomophthorales: Neozygitaceae) under controlled conditions: first observations at the population level

Virulence of entomopathogens is often measured at the individual level using a single host individual or a group of host individuals. To what extent these virulence assessments reflect the impact of an entomopathogen on their host in the field remains largely untested, however. A methodology was developed to induce epizootics of the cassava green mite fungal pathogen Neozygites tanajoae under controlled conditions to evaluate population-level virulence of two (one Beninese and one Brazilian) isolates of the entomopathogen—which had shown similar individual-level virulence but different field impacts. In unrepeated separate experiments we inoculated mite-infested potted cassava plants with either 50 or 25 live mites (high and low inoculum) previously exposed to spores of N. tanajoae and monitored the development of fungal infections for each isolate under the same conditions. Both isolates caused mite infections and an associated decline in host mite populations relative to the control (without fungus) in all experiments, but prevalence of the fungus varied with isolate and increased with inoculum density. Peak infection levels were 90% for the Beninese isolate and 36% for the Brazilian isolate at high inoculum density, and respectively 17% and 25% at low inoculum density. We also measured dispersal from inoculated plants and found that spore dispersal increased with host infection levels, independent of host densities, whereas mite dispersal varied between isolates. These results demonstrate that epizootiology of N. tanajoae can be studied under controlled conditions and suggest that virulence tests at the population level may help to better predict performance of fungal isolates than individual-level tests.     

Morphological, Biochemical and Molecular Approaches for Comparing Typical and Atypical Paracoccidioides brasiliensis Strains

We evaluated the morphology of typical and atypical Paracoccidioides brasiliensis strains and the expression of its 43 kDa glycoprotein (GP43). Strains of P. brasiliensis preserved under mineral oil for long periods of time presented different morphological patterns on peptone, yeast-extract and glucose (PYG) agar. The intravenous inoculation in BALB/c mice confirmed that a strain bearing morphological alterations was non-virulent. In contrast, another strain also maintained under mineral oil but which did not exhibit such morphological dysfunction was as virulent as the well characterized Pb 339 and Pb 18 strains. The expression of the main antigen expressed by P. brasiliensis, GP43, was assessed in culture filtrates by western immunoblots. Typical and atypical strains were capable of secreting the glycoprotein, except strain Pb IOC 1059. The identity of the atypical strains was confirmed by PCR using specific primers for gp43, though the single PCR-fragment varied in size for the atypical strains. The PCR fragments from an atypical strain, Pb IOC 1210, and the typical Pb 339 and Pb IOC 3698 strains were sequenced and blasted to the gp43 gene from the Pb 18 strain (GenBank AY005429). These results ensured the identity of the atypical strains as P. brasiliensis, and suggested a relationship between the alteration of morphological differentiation and the virulence factor following storage under mineral oil.     

<Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> Uses Endogenous and Exogenous Arachidonic Acid for PGE<Subscript>x</Subscript> Production

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent deep mycosis in Latin America. Production of eicosanoids during fungal infections plays a critical role on fungal biology as well as on host immune response modulation. The purpose of our study was to assess whether P. brasiliensis strains with different degree of virulence (Pb18, Pb265, Bt79, Pb192) produce prostaglandin E_x (PGE_x). Moreover, we asked if P. brasiliensis could use exogenous sources of arachidonic acid (AA), as well as metabolic pathways dependent on cyclooxygenase (COX) enzyme, as reported for mammalian cells. A possible association between this prostanoid and fungus viability was also assessed. Our results showed that all strains, independently of their virulence, produce high PGE_x levels on 4 h culture that were reduced after 8 h. However, in both culture times, higher prostanoid levels were detected after supplementation of medium with exogenous AA. Treatment with indomethacin, a COX inhibitor, induced a reduction on PGEx, as well as in fungus viability. The data provide evidence that P. brasiliensis produces prostaglandin-like molecules by metabolizing either endogenous or exogenous AA. Moreover, the results suggest the involvement of these mediators on fungal viability.     

Differences in Cell Morphometry,Cell Wall Topography and Gp70 Expression Correlate with the Virulence of Sporothrix brasiliensis Clinical Isolates

Sporotrichosis is a chronic infectious disease affecting both humans and animals. For many years, this subcutaneous mycosis had been attributed to a single etiological agent; however, it is now known that this taxon consists of a complex of at least four pathogenic species, including Sporothrix schenckii and Sporothrix brasiliensis. Gp70 was previously shown to be an important antigen and adhesin expressed on the fungal cell surface and may have a key role in immunomodulation and host response. The aim of this work was to study the virulence, morphometry, cell surface topology and gp70 expression of clinical isolates of S. brasiliensis compared with two reference strains of S. schenckii. Several clinical isolates related to severe human cases or associated with the Brazilian zoonotic outbreak of sporotrichosis were genotyped and clustered as S. brasiliensis. Interestingly, in a murine subcutaneous model of sporotrichosis, these isolates showed a higher virulence profile compared with S. schenckii. A single S. brasiliensis isolate from an HIV-positive patient not only showed lower virulence but also presented differences in cell morphometry, cell wall topography and abundant gp70 expression compared with the virulent isolates. In contrast, the highly virulent S. brasiliensis isolates showed reduced levels of cell wall gp70. These observations were confirmed by the topographical location of the gp70 antigen using immunoelectromicroscopy in both species. In addition, the gp70 molecule was sequenced and identified using mass spectrometry, and the sequenced peptides were aligned into predicted proteins using Blastp with the S. schenckii and S. brasiliensis genomes.     

Virulence and the Production of Endo-1,4-β-glucanase by Isolates of Alternaria alternata Involved in the Moldy-core Disease of Apples

Alternaria alternata, is the predominant fungal pathogen responsible for moldy‐core in apple cultivars of the Red Delicious group. Here we report on the association between virulence of natural isolates of A. alternata, their production of endo‐1,4‐β‐glucanase (EG) and moldy‐core development in apple fruits. Based on decay development following wound inoculations of mature fruits, three of 150 isolates, collected in three orchards in northern Israel and representing low, moderate and high virulence, were selected and designated Rm44, Er30 and Sh42, respectively. All three isolates secreted EG when grown on enzyme‐inducing medium (EIM) containing commercial cellulose or apple cell walls and this production was related to their degree of virulence. Polyacrylamide gel electrophoresis (PAGE) revealed quantitative differences between the three isolates, relative to their virulence. When fungal extracts were run in native gels, a single band with a molecular mass of 23 kDa showing EG activity was produced by the high‐ (Sh42) and the medium‐virulence (Er30) isolate but not by the low‐virulence (Rm44) isolate. A commercial cellulase preparation (containing endo‐ and exo‐1,4‐β‐glucanase) placed on pricked fruit led to the formation of symptoms similar to those developing on A. alternata‐inoculated fruits within 2–4 days. Inoculation of bloom clusters at full bloom with the highly virulent isolate (Sh42) of A. alternata resulted in a significantly higher infection in fruits (58%) than in those inoculated with the low‐virulence isolate (Rm44) (30%). Our results suggest that the moldy‐core symptoms caused by A. alternata in apple, could be related to the ability of the fungus to produce EG in developing lesions.     

Colletotrichum acutatum is the main cause of Colletotrichum leaf disease of rubber in Sri Lanka

Colletotrichum gloeosporoides has been described as the causal agent of Colletotrichum leaf disease of rubber in Sri Lanka and other parts of the world since 1905. A study carried out on vegetative and reproductive characteristics of 52 isolates from Colletotrichum leaf disease lesions on Hevea brasiliensis in Sri Lanka revealed that only 18 isolates belong to Colletotrichum gloeosporioides. The remaining 34 isolates represented C. aculatum indicating that C. acutatum is the main cause of Colletotrichum leaf disease in Sri Lanka.This revised version was published online in October 2005 with corrections to the Cover Date.     

Protective effect of prior immunization on ocular paracoccidioidomycosis in guinea pigs

The present study reproduced the experimental model of ocular paracoccidioidomycosis in guinea pigs, by the intracardiac inoculation of yeast-forms of P. brasiliensis. Ocular involvement was observed in 80% of the infected animals. The uvea, ciliary body, choroid, iris, lids and the conjunctiva were the structures most commonly affected. To protect the animals against the infection, an immunization protocol was standardized utilizing a P. brasiliensis soluble antigen in Freund's complete adjuvant, administered weekly, during 3 weeks, by the subcutaneous route. Two weeks later, previously immunized guinea pigs were challenged by the intracardiac route with yeast-forms of P. brasiliensis (vaccinated group). When compared with a control group (infection in the absence of prior immunization), the vaccinated animals developed higher levels of anti-P. brasiliensis cellular and humoral immune response and a three times lower frequency of ocular involvement (85.7% vs 28.5%). In addition, the ocular lesions were significantly more localized and contained less fungal cells. The data demonstrated that the subcutaneous immunization was effective in decreasing the frequency and extent of ocular lesions, as well as in blocking fungal multiplication.     

Dialysable leukocyte extracts modify the course of experimental paracoccidioidomycosis in the Syrian hamster

The effect of dialysable leukocyte extracts (DLE) obtained from hamsters immunized withParacoccidioides brasiliensis (immune DLE) and from non-immunized hamsters (non-immune DLE) was studied in hamsters inoculated withP. brasiliensis by the intratesticular route. Treatment with immune or non-immune DLE was started during the third week of infection and was repeated at 7, 11, 15 and 19 weeks. A group of untreated infected animals was used as control. Animals were submitted to the delayed hypersensitivity skin test toP. brasiliensis antigen (PbAg) in vivo and assayed in vitro by the macrophage migration inhibition test in the presence of Phytohemagglutinin (PHA) and PbAg and by immunodiffusion for specific antibody. The animals were sacrificed at 4, 8, 12, 16 and 20 weeks. The morphology and extension of the lesions were studied at the inoculation site, and in lymph nodes, lungs, liver, spleen and kidneys. In contrast to the controls, animals treated with both DLEs maintained a positive cell-mediated immune response throughout the experiment and developed less extensive infection with a significantly lower number of fungi in the lesions. The results suggest that immune and non-immune DLE preparations modified the evolution of experimental paracoccidioidomycosis with equal efficiency. This similarity may be explained by the immunoregulatory activities of both extracts.     

Pathogenicity of hyphomycetous fungi against <Emphasis Type="Italic">Cyclocephala signaticollis</Emphasis>

Susceptibility of the white grub Cyclocephala signaticollis Burmeister (Coleoptera: Scarabaeidae: Dynastinae) larvae to seven isolates of Beauveria bassiana (Balsamo) Vuillemin, five of Metarhizium anisopliae (Metschnikoff) Sorokin and two of Paecilomyces lilacinus (Thom) Samson (Deuteromycotina: Hyphomycetes) was investigated. Among 14 fungal isolates screened the most virulent was a B. bassiana isolate (Bb 53) that caused 70% mortality of third instar larvae in 40 days after inoculation at 1 × 10⁸ conida/ml. Strains of M. anisopliae and P. lilacinus showed low efficacy or no virulence to the target host.     

Alterations in the pathogenicity of oneParacoccidioides brasiliensis isolate do not correlative with itsin vitro growth

Thein vitro subcultivation of some microorganisms for long periods causes measurable loss of their pathogenicity, which can be reverted by reisolation from infected hosts. We compared the pathogenicity and thein vitro growth pattern of oneP. brasiliensis isolate (Pb 18) in its yeast phase, using the following samples: 1) The original pathogenic Pb 18 (OP). 2) Pb 18 attenuated by continuousin vitro subcultivation (AT). 3) Pb 18 (AT) reisolated from susceptible B 10.A mice (RS). 4) Pb 18 (AT) reisolated from resistant A/SN mice (RR). Pathogenicity was evaluated by anatomopathology and mortality of mice infected i.p. with 5×10⁶ fungi. Median survival times of mice infected with OP ranged from 74 to 117 days during the first 51 months of subculturing; with more cycles of subculturing the median survival time increased, reaching 250 days at the 64^th month. This indicated decreasing virulence of OP during this period of subculturing. Survival of mice infected with RS and RR was respectively 112 and 123 days, which is similar to the behavior of the OP variant. Thein vitro growth curve profile of RR showed significantly higher numbers of total and viable yeasts than the other studied variant. These results show that: 1) Pb 18 isolate loses its pathogenicity by continuous subcultivation. This phenomenon is reverted by reisolation from mice, independently from their susceptibility to the fungus; 2) thein vitro growth patterns of Pb 18 do not correlate with alterations in pathogenicity but are influenced by the host's environment.     

Characterisation of Isaria fumosorosea isolates and their virulence toward the Diamondback Moth,Plutella xylostella

Some morphological and physiological characteristics of an Isaria fumosorosea isolate with diminished virulence, IFCF01-D, and its parent isolate, IFCF01, were evaluated and laboratory bioassays were performed to assess their virulence against Plutella xylostella. The relationship among these traits and virulence against P. xylostella is discussed. There were no significant differences in conidial viability, spore production and the time required for 50% germination (GT₅₀). Spore viability after incubation for 24 h at 25°C was greater than 98% for both isolates tested. Spore production on potato dextrose agar after 14 days incubation at 25°C was 4.68 × 10⁸ and 4.59 × 10⁸ conidia/mL for IFCF01 and IFCF01-D, respectively. When exposed to high temperatures (40, 45, 50 or 55°C) through a water bath for 10 min, conidial germination ranged from 0.83% to 84.0% for IFCF01 and 0% to 86% for IFCF01-D. Germination rate showed a negative relationship with the exposure temperature for both isolates. The per cent germination of isolate IFCF01 24 h after ultraviolet (UV) radiation (18 W, 240–260 nm) varied from 0% to 92% and 0% to 81% for IFCF01-D. Germination rate and the exposure time exhibited a negative correlation for both isolates tested. Conidial surface hydrophobicity of IFCF01 (60%) was significantly higher than that of isolate IFCF01-D (53%). Subsequently, using the cicada exuviae as the substrate for enzymatic analysis, Pr1 and chitinase activity demonstrated the contrasting virulence traits: higher specific activities for the more virulent IFCF01 and lower enzymatic levels for isolate IFCF01-D.     

The impact of nutrition on spore yields for various fungal entomopathogens in liquid culture

Spore yields were measured for various fungal entomopathogens grown in six nutritionally different liquid media with low and high carbon concentrations (8 and 36 g l^–1, respectively) at carbon-to-nitrogen (C:N) ratios of 10:1, 30:1 and 50:1. Six fungi were tested: two Beauveria bassiana strains, three Paecilomyces fumosoroseus strains and one Metarhizium anisopliae strain. Spore yields were examined after 2, 4 or 7 days growth. In general, highest spore yields were obtained in media containing 36 g/l and a C:N ratio of 10:1. After 4 days growth, highest spore yields were measured in the three Paecilomyces isolates (6.9–9.7 × 10⁸ spores ml^–1). Spore production by the B. bassiana isolates was variable with one isolate producing high spore yields (12.2 × 10⁸ spores ml^–1) after 7 days growth. The M. anisopliae isolate produced low spore concentrations under all conditions tested. Using a commercial production protocol, a comparison of spore yields for the coffee berry borer P. fumosoroseus and a commercial B. bassiana isolate showed that highest spore concentrations (7.2 × 10⁸ spores ml^–1) were obtained with the P. fumosoroseus isolate 2-days post-inoculation. The ability of the P. fumosoroseus strain isolated from the coffee berry borer to rapidly produce high concentrations of spores prompted further testing to determine the desiccation tolerance of these spores. Desiccation studies showed that ca. 80% of the liquid culture produced P. fumosoroseus spores survived the air-drying process. The virulence of freshly produced, air-dried and freeze-dried coffee berry borer P. fumosoroseus blastospores preparations were tested against silverleaf whiteflies (Bemisia argentifolii). While all preparations infected and killed B. argentifolii, fresh and air-dried preparations were significantly more effective. These results suggest that screening potential fungal biopesticides for amenability to liquid culture spore production can aid in the identification of commercially viable isolates. In this study, P. fumosoroseus was shown to possess the production and stabilization attributes required for commercial development.