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PubMed1977

Effect of coumarin on growth and metabolism of Pythium.

Coumarin concentrations that inhibited growth of Pythium sp. rapidly decreased the rate of incorporation of [14C]glucose into the mycelium. Coumarin also reduced drastically the carbohydrate and protein content of the cytoplasm and, to a lesser extent, the amino acid content and cell wall fraction. Enzymes related to the metabolism of cell wall polysaccharides were not affected during early exposure to the inhibitor. The possible mechanism of coumarin action via inhibition of glucose uptake is discussed in the light of the present findings.

Amino Acids↗
PubMed1997

Evidence for signaling between the phytopathogenic fungus Pythium ultimum and Pseudomonas fluorescens F113: P. ultimum represses the expression of genes in P. fluorescens F113, resulting in altered ecological fitness.

There is increasing evidence that communication between members of the same species, as well as members of different species, is important for the survival of microorganisms in diverse ecological niches, such as the rhizosphere. To investigate whether the phytopathogen Pythium ultimum could alter gene expression in the biocontrol strain Pseudomonas fluorescens F113, which protects the roots of sugar beet from the fungus, a screening system was developed to detect differential expression of bacterial genes in the presence of P. ultimum. The transposon Tn5, containing a promoterless lacZ reporter gene, was used to generate a library of transcriptional gene fusions in P. fluorescens F113. By this screening procedure, five P. fluorescens F113 gene clusters were identified and shown to be repressed in the presence of P. ultimum. The ecological fitness of three of the five reporter mutants in the rhizosphere of seed-inoculated sugar beet was lower than that of the wild type. Furthermore, all five mutants were impaired in their ability to subsequently colonize the rhizosphere of uninoculated sugar beet sown repeatedly in the same soil. With the exception of reporter mutant SF10, which was impaired in nitrogen metabolism, the reporter mutants had growth requirements and biocontrol abilities similar to those of the wild type. This is the first reported case of a fungus repressing the expressing of bacterial genes.

Ecology↗
PubMed1992

Immunoblot analysis of the humoral immune response to Pythium insidiosum in horses with pythiosis.

Reactions to Pythium insidiosum by sera from horses with active pythiosis were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Five strains of P. insidiosum were grown in nutrient broth and then sonicated. After centrifugation, supernatant antigens were separated by SDS-PAGE. An exoantigen of Conidiobolus coronatus was also tested. Bands with molecular weights between 97,000 and 14,000 were identified by Coomassie blue and silver staining. After being transferred to nitrocellulose, the antigens were reacted against sera from six horses with pythiosis, sera from four horses cured a year earlier by vaccination, and sera from five healthy horses. The sera from horses with pythiosis recognized at least 20 antigens in all strains. Three antigens with molecular weights of 32,000, 30,000, and 28,000 appeared to be immunodominant and specific. Sera from horses cured by immunotherapy showed only five very weak bands, three of them the 32,000-molecular-weight (32K), 30K, and 28K antigens. No bands were observed with sera from healthy horses or sera from horses with a variety of other infections. Sera from horses with pythiosis cross-reacted with the 44K antigen of C. coronatus. The immunodominant antigens described here may be useful for diagnostic purposes and in immunotherapy for this oomycotic infection in horses.

Animals↗
PubMed1993

Life cycle of the human and animal oomycete pathogen Pythium insidiosum.

Pythium insidiosum, the etiologic agent of pythiosis insidiosii, causes life-threatening infections in humans and animals. Previous studies of the epidemiology of this disease hypothesized about the possible life cycle of this oomycete. Details, however, were not provided on the steps required to cause infection. We investigated the life cycle of P. insidiosum by inoculating pieces of equine skin and plant leaves and then studying the ensuing events with a scanning electron microscope. Our observations revealed that zoospores had a strong tropism for skin tissue, horse and human hair, and water lily and grass leaves and a weak attraction to a variety of other leaves. Encysted zoospores were observed on the favored leaves and skin. There they produced germ tubes and later abundant hyphal filaments that penetrated leaf tissues. Young sporangia had compact, thick walls. The sporangial wall was reduced to a fragile membrane when the sporangia had produced well-differentiated biflagellate zoospores. The encysted zoospores secreted an amorphous material that permitted the zoospores to adhere to skin and plant tissues. On the basis of these findings, a model to explain the life cycle of P. insidiosum is proposed.

Animals↗
PubMed2004

Identification of emerging human-pathogenic Pythium insidiosum by serological and molecular assay-based methods.

Pythium insidiosum is a pathogen that causes disease in both animals and humans. Human infection is rare; however, when it does occur, most patients, especially those having underlying hemoglobinopathy syndromes, such as thalassemia, exhibit a severe form. We identified four isolates of P. insidiosum. Two were recovered from tissue biopsy specimens from thalassemic and leukemic patients, one was derived from brain tissue from a thalassemic patient, and another was isolated from a corneal ulcer from a fourth patient. Western blotting and an enzyme-linked immunosorbent assay (ELISA) were performed with a serum sample derived from one thalassemic patient. The methods used to identify the P. insidiosum isolates were based on morphology, nucleic acid sequencing, and a PCR assay. To confirm the identification, portions of the 18S rRNA genes of these four isolates were sequenced. The sequences were shown to be homologous to previously described P. insidiosum DNA sequences. In addition, PCR amplification of the internal transcribed spacer region specific for P. insidiosum was positive for all four isolates. The ELISA with the serum sample from the thalassemic patient gave a positive result from a serum dilution of 1:800. Finally, Western immunoblotting with this serum sample showed positive immunoglobulin G recognition for proteins of 110, 73, 56, 42 to 35, 30 to 28, 26, and 23 kDa. The results of this study show that both molecularly based diagnostic and serodiagnostic techniques are useful for the rapid identification of human pythiosis. The predominant antigens recognized by Western blotting may be useful in the development of a more sensitive and specific diagnostic tool for this disease.

Antigens↗
PubMed2006

Identification of a novel 74-kiloDalton immunodominant antigen of Pythium insidiosum recognized by sera from human patients with pythiosis.

The oomycetous, fungus-like, aquatic organism Pythium insidiosum is the etiologic agent of pythiosis, a life-threatening infectious disease of humans and animals that has been increasingly reported from tropical, subtropical, and temperate countries. Human pythiosis is endemic in Thailand, and most patients present with arteritis, leading to limb amputation and/or death, or cornea ulcer, leading to enucleation. Diagnosis of pythiosis is time-consuming and difficult. Radical surgery is the main treatment for pythiosis because conventional antifungal drugs are ineffective. The aims of this study were to evaluate the use of Western blotting for diagnosis of human pythiosis, to identify specific immunodominant antigens of P. insidiosum, and to increase understanding of humoral immune responses against the pathogen. We performed Western blot analysis on 16 P. insidiosum isolates using 12 pythiosis serum samples. These specimens were derived from human patients with pythiosis who had different forms of infection and lived in different geographic areas throughout Thailand. We have identified a 74-kDa immunodominant antigen in all P. insidiosum isolates tested. The 74-kDa antigen was also recognized by sera from all patients with pythiosis but not by control sera from healthy individuals, patients with thalassemia, and patients with various infectious diseases, indicating that Western blot analysis could facilitate diagnosis of pythiosis. Therefore, the 74-kDa antigen is a potential target for developing rapid serodiagnostic tests as well as a therapeutic vaccine for pythiosis. These advances could lead to early diagnosis and effective treatment, crucial factors for better prognosis for patients with pythiosis.

Antibodies↗
PubMed1981

Changes in the chemical composition of oospores of Pythium aphanidermatum during bimodal germination.

The developmental mode by which oospores of Pythium aphanidermatum germinate may be controlled by the concentration of glucose present in the germination medium. When incubated in a liquid medium containing emulsified lecithin without added glucose (S + L), oospores germinated indirectly, with zoospore release occurring during the 10th to 13th h. A decrease in dry weight of the fungus took place by the 13th h of indirect germination. When 0.2 mM glucose was added to S + L, the dry weight of germlings increased and direct germination occurred without zoospore formation. Polar lipid comprised the bulk of total acylglycerides of dormant oospores. A greater decrease in total fatty acid content took place during direct germination than during indirect germination. During both developmental modes of germination, polar lipid content decreased considerably; neutral lipids, however, increased during indirect germination and decreased during direct germination. Protein content of the fungus increased in both developmental modes of germination, being initially 9.7% of the dry weight of dormant oospores. After 13 h of incubation, KOH-soluble polysaccharide decreased by 35% during direct germination and by 48% during indirect germination.

Fatty Acids↗
PubMed1996

A dual cofactor-specific isocitrate dehydrogenase from Pythium ultimum.

Isocitrate dehydrogenase is considered to be one of the key regulatory enzymes in the conversion of glucose into fatty acids by oleaginous microorganisms. A dual coenzyme-specific isocitrate dehydrogenase (EC 1.1.1.41) (IDH) was isolated from the primitive fungus Pythium ultimum and purified by 211-fold by sequential ion-exchange, affinity, and gel filtration chromatographies. Specific activity of the partially purified enzyme was 76.2 mumol/(min.mg protein) with NAD+ and 40% less active with NADP+. Optimum pH for activity was 8.5-9.5. K(m) values for threo-D-isocitrate and NAD+ were 0.031 and 0.55 mM, respectively. The estimated molecular mass of the IDH was 96 kDa under nondenaturing conditions and 48 kDa under denaturing conditions, suggesting that the enzyme is composed of two subunits of the same size. The enzyme was relatively stable up to 55 degrees C, but no activity was detected after exposure to 65 degrees C for 15 min. Mg2+ or Mn2+ were required for activity.

Cations, Divalent↗
PubMed2002

Evaluation of microbial culture techniques for the isolation of Pythium insidiosum from equine tissues.

The purpose of this study was to evaluate the effects of sample handling, storage, and culture techniques on the isolation of Pythium insidiosum from infected equine tissues. Tissue and kunker samples obtained immediately posteuthanasia from a horse with subcutaneous pythiosis were used to assess the effects of sample type (kunkers vs. tissues), media type (selective vs. nonselective), storage technique, and storage time on P. insidiosum isolation rate. Overall, isolation rates were higher from fresh kunkers (94.6%) and stored kunkers (76.4%) than from fresh tissues (8.3%) or stored tissues (4.6%). Isolation of P. insidiosum also occurred more often on antibiotic-containing media than on nonselective media for both fresh and stored samples. For samples that were stored for 1-3 days prior to culture, P. insidiosum isolation rates were highest for the following techniques: kunkers stored at room temperature and plated on selective media (100%), kunkers stored at 4 C and then plated on either nonselective (91.7%) or selective (95.8%) media, kunkers stored on cold packs and then plated on either nonselective (93.8%) or selective (100%) media, kunkers stored in ampicillin solution and plated on selective media (100%), and kunkers stored in ampicillin/gentocin solution and plated on selective media (87.5%). For samples stored for 4-5 days, P. insidiosum isolation rates were highest for kunkers stored at 4 C and then plated on either nonselective (81.3%) or selective (87.5%) media, kunkers stored in ampicillin solution and then plated on selective media (87.5%), and kunkers stored in ampicillin/gentocin solution and plated on selective media (87.5%). Results of this study suggest that optimal isolation rates of P. insidiosum from infected equine tissues are achieved by culturing fresh kunkers on selective media. For samples that cannot be processed immediately, acceptable handling techniques include storage at room temperature for up to 3 days, refrigeration for up to 5 days, shipping on cold packs, and storage in antibiotic solution, each combined with subsequent inoculation on selective media.

Animals↗
PubMed2004

Granulomatous pneumonia caused by Pythium insidiosum in a central American jaguar, Panthera onca.

A 7-month-old, male jaguar presented with dyspnea and leukocytosis unresponsive to antibiotic therapy. Radiographs revealed unilateral pulmonary consolidation. An exploratory thoracotomy was performed, and the left lung, which contained a large multilobular mass with extensive fibrosis and numerous caseonecrotic foci, was removed. Microscopically, eosinophilic granulomatous inflammation surrounded broad (4.4-8.3 microm) rarely septate hyphae. A diagnosis of Pythium insidiosum infection was confirmed by immunohistochemistry, immunoblot serology, culture, and polymerase chain reaction. Dyspnea recurred despite treatment, and the animal succumbed 3 weeks after surgery. Necropsy findings indicated that death resulted from occlusion of the right main stem bronchus by a fungal granuloma. The oomycete P. insidiosum typically causes granulomatous disease of the skin or gastrointestinal tract in animals and arteritis, keratitis, or cellulitis in humans. Infection is uncommon in felines, and pulmonary involvement is rare. This report details the first case of P. insidiosum infection in an exotic felid and provides the first description of primary pulmonary pythiosis in any species.

Animals↗
PubMed1989

Canine pythiosis--isolation and identification of Pythium insidiosum.

Pythium insidiosum was isolated from the subcutaneous tissue of a 1-year-old tan crossbreed dog and from the intestinal tract of an 18-month-old Samoyed male. Gomori's methenamine silver stain was superior to hematoxylin and eosin in demonstrating the organism in tissue sections. The agent was identified as P. insidiosum by zoospore formation in an aqueous yeast extract solution containing grass blades. Exoantigens produced in culture were shown to be identical to known P. insidiosum antigens by microimmunodiffusion.

Animals↗
PubMed2000

Control of Pythium root rot on hydroponically grown cucumbers with silver-coated cloth.

Silver-coated cloth (SCC) effectively controlled root rot that was caused by Pythium aphanidermatum in hydroponically grown cucumber plants. The presence of SCC in the hydroponic solution reduced the root rot from 100% to 10% 20 days after inoculation with zoospores of P. aphanidermatum. It was suggested that the inhibition of SCC was caused not only by the silver ion dissolved from SCC, but also by the metallic silver and silver compounds formed on the surface of the root.

Cucumis sativus↗
PubMed2002

Duodenal obstruction caused by infection with Pythium insidiosum in a 12-week-old puppy.

Pythium insidiosum is an aquatic fungus-like organism that causes a serious chronic granulomatous disease called pythiosis in animals and humans in tropical and subtropical regions of the world. In North America, pythiosis is most often diagnosed in the Gulf Coast states. Early recognition of the disease is crucial to successful treatment, which includes surgical resection of granulomatous lesions and administration of antifungal agents. Despite increasing availability of diagnostic tests, intestinal pythiosis is insidious and is often not detected until lesions are extensive. Intestinal pythiosis was diagnosed in a 12-week-old puppy from South Carolina examined because of vomiting, diarrhea, and anorexia. Pythiosis was not initially suspected because of the young age of the patient and because pythiosis is uncommon in this area.

Animals↗
PubMedDate not supplied

[Effect of exogenous sterols on the growth and fatty acid composition of the oomycete Pythium debaryanum].

Exogenous ergosterol and cholesterol were found to affect the growth and lipogenesis of the oomycete fungus Pythium debaryanum, which is unable to synthesize de novo steroid compounds. These sterols stimulated the growth of the fungus during its submerged cultivation in glucose-peptone medium. This was accompanied by the shortening of the lag phase, the lengthening of the period of active growth, and by a 3.7- or 4.3-fold increase in the maximum biomass in response to the addition of ergosterol or cholesterol, respectively. In the presence of ergosterol, the cellular content of polyenoic fatty acids increased, and the relative content of eicosapolyenoic fatty acids reached 31.4% of the total amount of fatty acids in cells. Conversely, cholesterol decreased the cellular content of polyenoic acids, and the relative content of eicosapolyenoic acids fell to 19.6% of the total amount of fatty acids. It may be inferred that exogenous sterols enhance the yield of pharmacologically active polyenoic acids because of the growth stimulation.

Fatty Acids↗
PubMedDate not supplied

[Effect of vitamin E and its functional analogs with different molecular structures on growth and lipids content of Pythium debaryanum].

The effect of exogenously added vitamin E and its synthetic analogues (the hydrophilic form of vitamin E and chromans C13 and C1) on the growth, lipogenic activity, and the fatty acid composition of the eicosapolyenoic acid-synthesizing oomycete Pythium debaryanum was studied. The effect was found to depend on the molecular structure of particular compounds. For instance, vitamin E and chroman C13 stimulated fungal growth, whereas chroman C1 inhibited it. The hydrophilic form of vitamin E enhanced the lipogenic activity of the oomycete. The studied compounds, which possess antioxidant activity, did not exert any noticeable effect on the level and the degree of the unsaturation of fungal lipids.

Lipid Metabolism↗
PubMed2001

A novel protein elicitor (PaNie) from Pythium aphanidermatum induces multiple defense responses in carrot, Arabidopsis, and tobacco.

A novel protein elicitor (PaNie(234)) from Pythium aphanidermatum (Edson) Fitzp. was purified, microsequenced, and the corresponding cDNA was cloned. The deduced amino acid sequence contains a putative eukaryotic secretion signal with a proteinase cleavage site. The heterologously expressed elicitor protein without the secretion signal of 21 amino acids (PaNie(213)) triggered programmed cell death and de novo formation of 4-hydroxybenzoic acid in cultured cells of carrot (Daucus carota). Programmed cell death was determined using the tetrazolium assay and DNA laddering. Infiltration of PaNie(213) into the intercellular space of leaves of Arabidopsis (Columbia-0, wild type) resulted in necroses and deposition of callose on the cell walls of spongy parenchyma cells surrounding the necrotic mesophyll cells. Necroses were also formed in tobacco (Nicotiana tabacum cv Wisconsin W38, wild type) and tomato (Lycopersicon esculentum Mill.) but not in maize (Zea mays), oat (Avena sativa), and Tradescantia zebrina (Bosse), indicating that monocotyledonous plants are unable to perceive the signal. The reactions observed after treatment with the purified PaNie(213) were identical to responses measured after treatment with a crude elicitor preparation from the culture medium of P. aphanidermatum, described previously. The availability of the pure protein offers the possibility to isolate the corresponding receptor and its connection to downstream signaling-inducing defense reactions.

Adaptation, Physiological↗
PubMedDate not supplied

Chilling resistances of isolates of Pythium ultimum var. ultimum from the Arctic and Temperate Zones.

Chilling resistances in moss pathogenic fungi, Pythium ultimum var. ultimum, from Longyearbyen, Svalbard (78 degree N, 15 degree E), located in the Arctic Zone and in the same isolates from Temperate Zone, were determined. Both strains had similar optimum growth temperatures. However, the strains from Svalbard could grow and survive at 0 - 5 degrees C. In addition, chilling treatment induced irregular mycelial morphology in the Arctic isolates. On the other hand, the isolates from Japan did not grow at temperatures below 5C and were destroyed after chilling stress (0 degree C for 3 days or at 4 degrees C for 1 week). The results suggested that isolates from Svalbard highly adapted to the severe spring condition in Polar environments.

Arctic Regions↗