A biochemical screening approach to putatively differentiate mammalian pathogenic Oomycota species in the clinical laboratory.

The report of four novel mammalian pathogenic species of the genus Lagenidium prompted us to study the use of biochemical assays to differentiate the Oomycota mammalian pathogens Pythium insidiosum and Lagenidium spp. We investigated the reaction of 23 Lagenidium and eight Pythium species in various biochemical assays. Because the morphological features of the Oomycota species are similar to those of species in the Entomophthoramycota and Mucormycota, five fungal species with coenocytic hyphae were also included. We found that mammalian and plant isolates of Pythium spp. all hydrolysed sucrose, but Lagenidium species and the fungal strains did not. In addition, both Pythium spp. and Lagenidium spp. were found to be maltose-positive, whereas fungal strains did not hydrolyse this sugar. The fungal species and thermo-sensitive Lagenidium giganteum and Lagenidium humanum were urease-negative, but the mammalian Lagenidium spp. and Pythium spp. hydrolysed urea within 24  h. These findings suggest these assays can be used for the presumptive differentiation of mammalian Oomycota species in the laboratory.

Efficacy of the Trichophyton ajelloi and Lagenidium giganteum metabolites against mosquitoes after flash chromatography.

Entomopathogens are significant natural enemies for mosquitoes. We have investigated the adulticidal efficacies of metabolites of Trichophyton ajelloi and Lagenidium giganteum against Culex quinquefasciatus and Aedes aegypti simultaneously. The T. ajelloi was grown on Sabouraud's dextrose broth medium at 25 ± 2°C and relative humidity at 75 ± 5% for 15 days. L. giganteum was grown in peptone yeast extract glucose broth at 25 ± 2°C and relative humidity 75 ± 5% for 15 days. The filtrations of metabolites have been made by using Whatman-1 filter paper then with the flash chromatograph. The bioassays were conducted as per the World Health Organization's methods and protocols (2006). In this significant investigations, the metabolites of T. ajelloi have been found highly susceptible against A. aegypti with LC(99)-7.24 ml after an exposure time of 24 h with a comparison, the LC(99)-66 ml was observed against C. quinquefasciatus after exposure of 24 h. Moreover, the L. giganteum metabolites have shown higher pathogenicity against C. quinquefasciatus with LC(99)-11.3 ml and A. aegypti with LC(99)-15.49 ml. Although, the efficacy in adults could be achieved with higher concentration can be significant also. Their adulticidal activities in different climatic zones are plausible with metabolites which have better LT values of T. ajelloi.

Effects of culture medium and formulation on the larvicidal activity of the mosquito pathogen Lagenidium giganteum (Oomycetes: Lagenidiales) against Aedes aegypti.

In this work, we examined the production of infective zoospores of Lagenidium giganteum in four culture media, and the larvicidal activity of the cultures was determined against Aedes aegypti larvae, as well as the effect of polymer encapsulation. Medium containing sunflower seed extract showed the greatest production of zoospores, 5.92×10(6) zoospores/ml after six days of fermentation at 25±2°C and 150rpm shaking. This culture tested against A. aegypti 1st stage larvae caused different mortality rates at 24, 48 and 72h posttreatment. The LC(50) obtained was 43.9, 41.1 and 42.9µl of total culture/ml, at 24, 48 and 72h posttreatment respectively, while the culture grown in medium with soybean meal showed 3-5 times higher LC(50) values. Finally, the total culture including mycelium, zoospores and presporangia formulated with 2.5% pectin showed significantly higher mortality rates, around 100% more than the unformulated culture, whose values were from 40 to 1% at 3, 6, 9, and 12d posttreatment in the bioassays carried out in the laboratory to determine residual activity.

In vitro susceptibility of Pythium insidiosum and a Lagenidium sp to itraconazole, posaconazole, voriconazole, terbinafine, caspofungin, and mefenoxam.

OBJECTIVE: To evaluate the radial growth assay for use in in vitro susceptibility testing of Pythium insidiosum and a Lagenidium sp and to assess susceptibility of representative isolates to itraconazole, posaconazole, voriconazole, terbinafine, caspofungin, and mefenoxam. SAMPLE POPULATION: 6 isolates each of P insidiosum and Lagenidium sp. PROCEDURES: Isolates were plated in triplicate onto agar supplemented with antifungal compounds at concentrations of 0.025 to 8 microg/mL. Isolates on dimethyl sulfoxide- and water-supplemented agar served as control samples. Effect of antifungal concentration on colony diameter was assessed with a mixed linear model. Assay variability was assessed with the coefficient of variation. RESULTS: Colony growth was uniform (mean intra-assay and interassay coefficients of variation were < 5%). Minimal inhibition was evident with voriconazole and posaconazole at 8 microg/mL. Terbinafine at 8 microg/mL significantly reduced growth of P insidiosum and at > or = 1 microg/mL significantly reduced growth of the Lagenidium sp. Caspofungin and mefenoxam (concentrations > or = 1 microg/mL and > or = 0.025 microg/mL, respectively) significantly reduced growth of both pathogens. Mefenoxam at 0.1 microg/mL caused > 50% growth inhibition in 11 of 12 isolates and at 1 microg/mL caused > 90% inhibition in all isolates. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that the radial growth assay was a simple, reproducible technique for susceptibility testing of P insidiosum and a Lagenidium sp. Azoles had limited activity, whereas terbinafine and caspofungin caused significant but minimal to moderate inhibition. Only mefenoxam had a profound effect on both pathogens at concentrations likely to be achievable in tissues.

Efficacy of Lagenidium giganteum metabolites on mosquito larvae with reference to nontarget organisms.

Lagenidium giganteum is a water mold and an effective mosquito control agent with limited use due to poor survival and contamination during storage. Invert extracellular metabolites of L. giganteum is easy to produce, long shelf life, and a potential candidate in tropical climates. This fungus was grown in PYG broth in the laboratory at 25 +/- 2 degrees C, and relative humidity was maintained at 75 +/- 5% for 15 +/- 2 days. Filtration process of metabolites was done using Whatman filter paper, column chromatograph, and range syringe filters techniques. Then 5-ml fractions were collected and used to assay larvicidal efficacies. Larvicidal efficacies were performed against Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti at five different concentrations, viz. 1.68, 1.99, 2.17, 2.30, and 2.40 ppm. And also, filtrates were assessed against four species of nontarget organisms named Daphnia pulex, Cyclopes, Lymnea auriculeta, and tadpoles of Rana tigrina with different concentrations. The mortality values were subjected by the Probit analysis. The complete mortalities that resulted from applying filtrates dosage on all instars of mosquitoes persisted for a period of 24, 48, and 72 h, respectively. The efficacies in killing instar of three important vectors and safer for nontarget organisms with good biological stability of extracellular metabolites make this a promising alternative to mycelium and conidial-based larvicides. It could be regarded as fungal-based natural larvicide for the use of vector control.

The kinetics of Lagenidium giganteum growth in liquid and solid cultures.

AIMS: Production of the mosquito biolarvacide Lagenidium giganteum in solid culture has been proposed as an economic alternative to production in liquid culture because of observations of improved shelf life and efficacy upon storage. Understanding the differences between these production systems and estimating growth rate in solid culture are important for commercialization. In order to address these needs a logistic model was developed to describe the growth kinetics of L. giganteum produced in solid and liquid cultures. METHODS AND RESULTS: Kinetic parameters in the logistic model were estimated by nonlinear regression of CO2 evolution rate (CER) and biomass data from solid and liquid cultivation experiments. Lagenidium giganteum biomass was measured using DNA extracted directly from samples. The logistic model was fit to experimental biomass and CER data with low standard errors for parameter estimates. The model was validated in two independent experiments by examining prediction of biomass using on-line CER measurements. CONCLUSIONS: There were significant differences between maximum biomass density, maintenance coefficients, and specific growth rates for liquid and solid cultures. The maximum biomass density (mg dw ml-1) was 11 times greater for solid cultivation compared with liquid cultivation of L. giganteum; however, the maintenance coefficient (mg CO2 h-1 (mg dw)-1) was six times greater for liquid cultivation than in solid cultivation. The specific growth rate at 30 degrees C was approximately 30% greater in liquid cultivation compared with solid cultivation. Slower depletion of substrate and lower endogenous metabolism may explain the longer shelf life of L. giganteum produced in solid culture. SIGNIFICANCE AND IMPACT OF THE STUDY: A simple logistic model was developed which allows real-time estimation of L. giganteum biomass from on-line CER measurements. Parameter estimates for liquid and solid cultivation models also elucidated observations of longer shelf life for production in solid culture.