The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities.
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The entomopathogenic fungus Neozygites parvispora (Entomophthorales: Zygomycetes) grows in vitro as irregularly rod-shaped hyphal bodies in a complex medium. In order to simplify the medium composition and determine growth-promoting compounds for the cultivation of this fungus, we were looking for a rapid and quantitative method to estimate the number of living cells in small volumes of liquid culture. A colorimetric method for the determination of cell densities using MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] proved to be more accurate and timesaving than conventional hemocytometer counting. (+info)
Gastrointestinal zygomycotic infection caused by Basidiobolus ranarum: case report and review.
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Basidiobolus species are filamentous fungi belonging to the order Entomophthorales. Unlike other zygomycetes, Basidiobolus species have been mainly associated with a tropical form of subcutaneous zygomycosis in otherwise healthy individuals. Visceral disease caused by this pathogen is rare, but cases of gastrointestinal infection with Basidiobolus ranarum have been reported worldwide. In many of these reports, the inflammatory disease of the colon has been confused with Crohn's disease. We report the third case of B. ranarum gastrointestinal infection in the United States, which was initially treated as inflammatory bowel disease. (+info)
Pathology and epizootiology of Entomophaga maimaiga infections in forest Lepidoptera.
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The insect-pathogenic fungal pathogen Entomophaga maimaiga is endemic to northeastern Asia and was first found in North America in 1989. Due to repeated epizootics and spread within populations of the major forest defoliator in northeastern North America, the gypsy moth (Lymantria dispar), this pathogen has gained much notoriety. Although this pathogen was purposely introduced to North America for biological control of L. dispar in 1910 to 1911, it is questionable whether it became established at the time of release and then remained at innocuous levels until relatively recently. Alternatively, the fungal strain present in North America today could be a more recent accidental introduction. DNA analysis demonstrates that this pathogen differs significantly from North American members of the same species complex (the Lepidoptera-specific Entomophaga aulicae species complex), and, to date, isolates of this introduced pathogen display little heterogeneity in North America. Nonsusceptible lepidopteran larvae have been identified, and either E. maimaiga is unable to penetrate the cuticle or the fungus cannot survive within the hemocoel. In the latter case, although E. maimaiga grows as protoplasts lacking cell walls in the host hemolymph, glycoproteins on plasma membranes of the protoplasts could lead to host recognition. Epizootiological studies demonstrate a clear association between fungal activity and environmental moisture but little association with host density under hypothesized conditions of high fungal density. Prediction of the occurrence of epizootics is not yet possible. E. maimaiga is easily established in new areas by releasing azygospores, but the ability to use this pathogen further for biological control will depend, in large part, on the development of mass production systems. (+info)
In-vitro antifungal susceptibilities of Basidiobolus and Conidiobolus spp. strains.
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The in-vitro antifungal susceptibilities of nine isolates belonging to Basidiobolus spp. and seven to Conidiobolus spp. against six antifungals (amphotericin B, ketoconazole, miconazole, itraconazole, fluconazole and flucytosine) were tested. A broth microdilution method, generally following the NCCLS guidelines, was used. Inoculum concentrations of the order of 100 cfu/mL were obtained by culturing fungi in a broth medium (Czapeck broth supplemented with 2% Tween 80 and 0.07% agar). MICs and MFCs were highly variable and isolate-dependent, with the exception of those of flucytosine which were constantly very high. In general, however, Basidiobolus spp. displayed low MICs of fluconazole, itraconazole, ketoconazole and miconazole, and Conidiobolus spp. were resistant to all antifungals tested. (+info)
Purification and characterization of the single-strand-specific and guanylic-acid-preferential deoxyribonuclease activity of the extracellular nuclease from Basidiobolus haptosporus.
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An extracellular nuclease from Basidiobolus haptosporus (designated as nuclease Bh1) was purified to homogeneity by ammonium sulfate precipitation, heat treatment, negative adsorption on DEAE-cellulose, and chromatography on phenyl-Sepharose followed by FPLC on phenyl-Superose. The overall yield was 26%. The Mr of the purified enzyme, determined by gel filtration, was 41 000 whereas by SDS/PAGE (after deglycosylation) it was 30 000. It is a glycoprotein with a pI of 6.8. The optimum pH and temperature for DNA hydrolysis were 8. 5 and 60 degrees C, respectively. Nuclease Bh1 is a metalloprotein but has no obligate requirement for metal ions to be active, nor is its activity stimulated in the presence of metal ions. The enzyme was inhibited by Zn2+, Ag2+, Hg2+, Fe3+ and Al3+, inorganic phosphate, pyrophosphate, dithiothreitol, 2-mercaptoethanol, NaCl and KCl. It was stable to high concentrations of organic solvents and urea but susceptible to low concentrations of SDS and guanidine hydrochloride. Nuclease Bh1 is a multifunctional enzyme and its substrate specificity is in the order of ssDNA approximately 3'AMP >> RNA > dsDNA. Studies on its mode of action showed that it cleaved supercoiled pUC 18 DNA and phage M13 DNA, endonucleolytically, generating single base nicks. The enzyme hydrolyzed DNA with preferential liberation of 5'dGMP, suggesting it to be a guanylic acid preferential endoexonuclease. 5'dGMP, the end product of hydrolysis, was a competitive inhibitor of the enzyme. The absence of 5'dCMP as a hydrolytic product, coupled with the resistance of (dC)10 and deoxyribodinucleoside monophosphates having cytosine either at the 3' or the 5' end, indicates that C-linkages are resistant to cleavage by nuclease Bh1. (+info)
Gastrointestinal basidiobolomycosis in Arizona: clinical and epidemiological characteristics and review of the literature.
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Gastrointestinal basidiobolomycosis (GIB) is an unusual fungal infection that is rarely reported in the medical literature. From April 1994 through May 1999, 7 cases of GIB occurred in Arizona, 4 from December 1998 through May 1999. We reviewed the clinical characteristics of the patients and conducted a case-control study to generate hypotheses about potential risk factors. All patients had histopathologic signs characteristic of basidiobolomycosis. Five patients were male (median age, 52 years; range, 37--59 years) and had a history of diabetes mellitus (in 3 patients), peptic ulcer disease (in 2), or pica (in 1). All patients underwent partial or complete surgical resection of the infected portions of their gastrointestinal tracts, and all received itraconazole postoperatively for a median of 10 months (range, 3--19 months). Potential risk factors included prior ranitidine use and longer residence in Arizona. GIB is a newly emerging infection that causes substantial morbidity and diagnostic confusion. Further studies are needed to better define its risk factors and treatment. (+info)
Basidiobolus ranarum as an etiologic agent of gastrointestinal zygomycosis.
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Basidiobolus ranarum is a known cause of subcutaneous zygomycosis. Recently, its etiologic role in gastrointestinal infections has been increasingly recognized. While the clinical presentation of the subcutaneous disease is quite characteristic and the disease is easy to diagnose, gastrointestinal basidiobolomycosis poses diagnostic difficulties; its clinical presentation is nonspecific, there are no identifiable risk factors, and all age groups are susceptible. The case of gastrointestinal basidiobolomycosis described in the present report occurred in a 41-year-old Indian male who had a history of repair of a left inguinal hernia 2 years earlier and who is native to the southern part of India, where the subcutaneous form of the disease is indigenous. Diagnosis is based on the isolation of B. ranarum from cultures of urine and demonstration of broad, sparsely septate hyphal elements in histopathologic sections of the colon, with characteristic eosinophilic infiltration and the Splendore-Hoeppli phenomenon. The titers of both immunoglobulin G (IgG) and IgM antibodies to locally produced antigen of the fungus were elevated. The patient failed to respond to 8 weeks of amphotericin B therapy, and the isolate was later found to be resistant to amphotericin B, itraconazole, fluconazole, and flucytosine but susceptible to ketoconazole and miconazole. One other noteworthy feature of the fungus was that the patient's serum showed raised levels of Th2-type cytokines (interleukins 4 and 10) and tumor necrosis factor alpha. The present report underscores the need to consider gastrointestinal basidiobolomycosis in the differential diagnosis of inflammatory bowel diseases and suggests that, perhaps, more time should be invested in developing standardized serologic reagents that can be used as part of a less invasive means of diagnosis of the disease. (+info)
Disseminated basidiobolomycosis in an immunocompetent woman.
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Basidiobolomycosis is a chronic subcutaneous infection of the trunk and limbs due to Basidiobolus ranarum. The disease is well known in tropical areas, although recent cases of gastrointestinal basidiobolomycosis have also been reported in Arizona. We describe a young immunocompetent women who had presented with eosinophilia and lung infiltrates. She subsequently died, and diagnosis of disseminated basidiobolomycosis was made on the basis of histological features at autopsy. (+info)