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Conidiobolomycosis is an infection for which there are no sufficient clinical or in vitro data to support a consensus about the optimal treatment, and consequently, cases of therapeutic success and failure have been described for all forms of therapy ([1][1][–][2][3][3]). We have determined the in

The present study investigated the in vitro and the in vivo interactions among azithromycin, clarithromycin, minocycline, and tigecycline against Pythium insidiosum . In vitro antimicrobial activities were determined by the broth microdilution method in accordance with CLSI document M38-A2, and the antibiotic interactions were assayed using the checkerboard MIC format. In vivo efficacy was determined using a rabbit infection model. The geometric mean MICs of azithromycin, clarithromycin, minocycline, and tigecycline against P. insidiosum were, respectively, 1.91, 1.38, 0.91, and 0.79 μg/ml. By checkerboard testing, all combinations resulted in in vitro synergistic interactions (>60%). Antagonism was not observed. The in vivo studies showed that azithromycin (20 mg/kg/day twice daily) alone or in combination with minocycline (10 mg/kg/day twice daily) significantly decreased the fungal burden. This study demonstrates that azithromycin possesses potent curative efficacy against subcutaneous pythiosis in the rabbit model.

This paper reports pythiosis in a sheep from southwestern Paraná, Brazil, confirmed by indirect ELISA (Enzime-Linked Immunosorbent Assay) and immunohistochemistry, as well as it describes the macro and microscopic injuries, in order to understand the pathogenicity. A 4-year-old ewe from a flock of 30 Santa Inês sheep, raised semi-extensively with access to a weir, showed cachexia, bilateral enlargement in nasal region, a serous and bloody secretion with a fetid odor from its nose and swollen submandibular and retropharyngeal lymph nodes. Blood collection was performed trough jugular vein puncture in order to make complete blood cell count (CBC) and to obtain serum for the subsequent serological examination. As the hematological counts were within the normal range for sheep, the animal was euthanized and submitted to necropsy. Indirect ELISA resulted positive for pythiosis. Necropsy revealed necrosis of the hard palate with a diameter of 3.5cm and extending up to the nasal cavity, forming a fistula. Submandibular and retropharyngeal lymph nodes were enlarged and edematous on section. Microscopic findings for submandibular and retropharyngeal lymph nodes consisted in moderate infiltration of eosinophils mainly in the subcapsular sinus, characterizing reactive eosinophilic lymphadenitis. The nasal cavity revealed rhinitis and oral cavity stomatitis with necro-eosinophilic and pronounced multifocal granulomatous infiltration and presence of hyphae. Hyphae found in palate and nasal cavity were positive for Pythium insidiosum by Grocott's method and immunohistochemistry, the last one considered to be confirmatory for the pathogen diagnostic. This report has an important epidemiological aspect, as it is the first case of pythiosis in sheep confirmed by serology in South Brazil and an alert of possible infection by the pathogen in floodplains.

Pythiosis is a severe disease caused by Pythium insidiosum. Currently, the research on the treatment of pythiosis uses rabbits as an experimental infection model. To reduce the use of animals in scientific experimentation, alternative models are increasingly necessary options. The objective of this study was to establish a new experimental infection model for pythiosis using embryonated chicken eggs. First, we tested the inoculation of four zoospore concentrations into the egg allantoic cavity at three embryonic days. We observed that increased zoospore concentration causes a decrease in survival time, and at a later embryonic day (the 14th) of infection, embryos showed delayed mortality. To confirm the reproducibility of the model, we chose the 14th embryonic day for the inoculation of 50 zoospores/egg, and the experiment was repeated twice. Mortality began with 30% embryos 48 h after inoculation, and 95% embryos died within 72 h. There was no mortality in the uninfected control group. The infection was confirmed by culture, PCR and histopathology. Immunohistochemistry confirmed the presence of hyphae in blood vessels in the umbilical cords in 95% of embryos and only one liver (5%). Our results suggest that embryonated eggs can be a very useful alternative infection model to study pythiosis. This article is protected by copyright. All rights reserved.

Pythiosis is a severe and life-threatening disease that affects humans and various animal species. We report a model of vascular/disseminated pythiosis occurring after subcutaneous inoculation of 2 x 104 Pythium insidiosum zoospores/mL in immunocompromised BALB/c mice. For this model, we carried out two rounds of experiments. First, we evaluated two protocols of immunosuppression before inoculation: cyclophosphamide at 150 mg/kg (CYP group) and cyclophosphamide 200 mg/kg plus hydrocortisone acetate at 250 mg/kg (CYP+HCA group). It was not possible to obtain mortality in the CYP group; however, the combination of CYP+HCA altered disease outcomes, with mortality rates reaching 60%. Second, we used the CYP+HCA immunosuppression protocol to analyze the histological and immunological statuses triggered by disease. When we inoculated immunocompetent mice with P. insidiosum zoospores, self-healing occurred via increased levels of IL-2, IFN-γ and IL-17A, which are characteristic of the Th1/Th17 cytokine response. For infected and immunosuppressed mice, the cytokine profiles showed high levels of IL-10, IL-6 and TNF-α. Increased IL-10 values are related to fungal infection susceptibility and led us to speculate that infection may be established through suppression of the host immune response. In addition, histopathological evaluation of the kidneys and liver demonstrated the presence of hyphae and the cellular findings suggested an acute vascular inflammation that mimics vascular/disseminated pythiosis in humans. This is the first murine model for pythiosis that is useful both for understanding the pathogenesis of this disease and for evaluating new treatment approaches.

We describe here the in vitro activities of azithromycin, clarithromycin, minocycline, or tigecycline alone and in combination with amphotericin B, itraconazole, terbinafine, voriconazole, anidulafungin, caspofungin, or micafungin against 30 isolates of the oomycete Pythium insidiosum . The assays were based on the CLSI M38-A2 technique and the checkerboard microdilution method. The main synergisms observed were through the combination of minocycline with amphotericin B (73.33%), itraconazole (70%), and micafungin (70%) and of clarithromycin with micafungin (73.33%).

Pythiosis is a life-threatening infectious disease caused by the pathogenic oomycete Pythium insidiosum. This study is the first to evaluate the P. insidiosum glucan content and its biological activities. The enzymatic quantification of the glucans in P. insidiosum mycelia showed that the β-glucan content was 18.99% ± 3.59. The cell wall polysaccharide extract consisted of ∼81.7% carbohydrates (exclusively glucose) and ∼18.3% residual amino acids and peptides. The results from monosaccharide composition, methylation and 1D/2D NMR spectroscopy analyses indicated the presence of a highly branched (1,3)(1,6)-β- d -glucan, with (1,6)-β- d -glucopyranosil side-branching unit on average every 1–2 repeat units. In vitro, the β- d -glucan extract could significantly promote spleen lymphocyte proliferation in human, equine and mouse cell cultures. BALB/c mice that were subcutaneously pre-immunized with three doses of 0.5, 2.5 and 5.0 mg of β-glucan/mouse, showed a significant increase in IL-2, IL-6, IL-10, TNF-α and IL-17A production compared to non-immunized mice. These results suggested that β- d -glucan extract induces significant and specific Th17 cellular immune response and provided the theoretical basis for further experiments.

The fallow period can represent an opportunity to farmers if the field can be trimmed to receive finishing cattle. Nematode control remains important at this stage, and the use of biological agents such as nematophagous fungi is desirable. Nonetheless, the indiscriminate use of fungicides on agricultural crops may affect the development and survival of fungal propagules. Therefore, we evaluated the in vitro activity of 14 agricultural fungicides against the Duddingtonia flagrans strain ARSEF 5701. The assays were based on CLSI M38-A2 broth microdilution and the agar diffusion method. The fungicides azoxystrobin, cyproconazole, cyprodinil and tebuconazole showed minimum inhibitory concentrations for D. flagrans below their respective maximum residue limits. Although complete radial growth inhibition was only observed for carbendazim at the maximum concentration tested, D. flagrans proved to be resistant to only two fungicides, cyprodinil and iprodione. The present study indicates that D. flagrans is sensitive to most of the common agricultural fungicides, and resistance depends upon the structure, i.e., hypha or chlamydospore. On the basis of these in vitro results, we concluded that the use of agricultural fungicides could seriously hamper the survival of the fungus D. flagrans in amended soils.

a b s t r a c t Background: Pythium insidiosum is an oomycete classified in the kingdom Stramenopila. P. insidiosum hyphae are not able to initiate infection without the secretion of hydrolytic enzymes, which are consid- ered an important factor in microbial virulence. Aims: To evaluate the extracellular enzymatic activity of 14 Brazilian P. insidiosum isolates and a standard strain (ATCC 58637) by the API-ZYM System screening method. Methods: Zoospores were grown in RPMI 1640 broth, and 65 L of the liquid phase were inoculated in each cupule of the API-ZYM strips. Results: Differences in the enzymatic activities were observed among the isolates, although phosphohy- drolases and ester hydrolases were conspicuous among all isolates. -glucosidase was also present in most of the isolates. Enzymatic activities of -glucosidase and chymotrypsin were not observed, differing from a previous study involving Australian isolates and intracellular enzymes. Conclusions: The discrepancy in the enzymatic profile observed among Brazilian P. insidiosum isolates reflects the phenotypic variations found in susceptibility tests.

A cutaneousPythium insidiosumreinfection was diagnosed in an equine in Brazil. Lesions with focal presentation appeared 2 years apart. The first infection and even immunotherapy were not likely to develop enough immune response to prevent reinfection. The use of adjuvants should be considered in the immunotherapy of pythiosis.