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Pythium insidiosum is an important oomycete pathogen of mammals that causes pythiosis, an endemic disease in warm climates that stands out for its unfavorable prognosis, lethality in the affected species, and difficulties in treatment. This study evaluated in vitro anti-P. insidiosum potential of aqueous, hydroethanolic, and ethanolic extracts of indigenous wild mushrooms from southern Brazil. The extracts were prepared from Amanita gemmata, Amanita muscaria, Auricularia auricula, Gymnopilus junonius, Lactarius deliciosus, Laccaria laccata, Psilocybe cubensis, and Russula xerampelina. In vitro susceptibility assays employed the microdilution technique according to the M38-A2 protocol CLSI. The hydroethanolic and ethanolic extracts of R. xerampelina showed anti-P. insidiosum activity at minimum inhibitory concentrations ranging from 1.87--7.50 mg/mL. The other mushroom species extracts showed no inhibitory effects on growth of P. insidiosum. This is the first study to evaluate the antimicrobial activity of mushrooms on oomycetes, evidencing the antimicrobial potential of R. xerampelina on the pathogen P. insidiosum. So, the present study expands new perspectives, since the secondary metabolites produced by mushrooms can be potential targets for the development of new categories of medicines. However, considering the wide biodiversity of Brazilian mushrooms, we suggested that the search for other basidiomycetes species with anti-P. insidiosum action needs to be expanded. [ABSTRACT FROM AUTHOR]

ABSTRACT: This study evaluated the inhibitory effect of ozone gas (O3) on the mycelia and zoospores of Pythium insidiosum and verified the morphological changes it caused on the hyphae. The effects of O3 were evaluated on the mycelia (n = 21) and zoospores (n = 10) of P. insidiosum. O3 was bubbled into vials with sterile distilled water and mycelium or induction medium and zoospores. After 15 minutes, aliquots of mycelia and zoospores were grown on yeast agar at 37 °C/96 hours. Ozonated flasks were incubated at 37 ºC/72 hours; and every 24 hours, aliquots were subcultured at 37 ºC/96 hours. Transmission (TEM) and scanning (SEM) electron microscopy of O3-exposed hyphae were performed. The results revealed that O3 inhibited microorganism growth in all ozonated suspensions. Conversely, in control suspensions, P. insidiosum growth occurred within 24 hours of incubation. In the SEM, the treated hyphae presented cavitations and cell wall continuity loss; in the TEM, the organelles and cytoplasmic membrane disappeared, and the cell wall was retracted. We demonstrated, for the first time, that O3 causes irreversible damage on P. insidiosum hyphae and prove in vitro oomicidal action on zoospores and hyphae.

The aquatic oomycete Pythium insidiosum is an emerging pathogen highly relevant in human and veterinary medicine and an etiologic agent of pythiosis, a disease of worldwide distribution mainly affecting horses, dogs, and humans, presenting cutaneous, subcutaneous, ocular, gastrointestinal, and systemic forms. The available therapeutic methods to treat this disease and its forms are not entirely effective, thus highlighting the need to investigate the forms of treatments with better efficacy, such as compounds from different pharmacological classes, compounds of natural origin, and new technological alternatives, including nanotechnology. Therefore, this study evaluated scientific publications regarding the use of nanotechnology in P. insidiosum treatment. For this, a systematic literature review, was carried out on articles published from 2010 to 2022 on the LILACS, MEDLINE, Google Scholar, PubMed, and SciELO databases using the descriptors ‘Pythium insidiosum,’ ‘pythiosis,’ ‘nanotechnology,’ ‘nanoparticles,’ ‘nanoemulsion,’ and ‘treatment.’ We reported 162 articles for the researched theme; although, only four studies were included because they met the criteria established herein. A meta-analysis was used for the statistical analysis of the data obtained in vitro studies, and we reported the use of nanotechnology can be a promising alternative in developing antimicrobial compounds with anti-P. insidiosum activity. Nevertheless, additional research is needed to verify the potential use of this technology in clinical therapy against P. insidiosum infections. [ABSTRACT FROM AUTHOR]

A case of meningoencephalitis caused by Pythium insidiosum secondary to rhinitis is reported in a three-year-old crossbred sheep from a herd of 15 animals, raised extensively and with free access to a weir. The animal presented mild dyspnea, blindness, mydriasis, opisthotonos, nystagmus, incoordination, decreased mandibular tone, and spasticity of the pelvic limbs. Macroscopic examination of the nasal cavity showed a blackish-red, irregular, friable mass that bilaterally compromised the nasal septum and the rostral portion of the nasal turbinates. In the brain, there was diffuse thickening of the leptomeninges of the cerebellum and ventral portion of the brainstem characterized by yellowish, granular material associated with vessel hyperemia. On the floor of the fourth ventricle, there was deposition of yellowish, irregular, slightly granular material that protruded towards the obex and displaced the cerebellum dorsolaterally. Microscopically, there were pyogranulomatous, eosinophilic, necrotizing rhinitis and fibrinosuppurative, eosinophilic, necrotizing meningoencephalitis, both associated with thrombosis, vasculitis, and intralesional hyphae. The hyphae were impregnated with silver and presented thin, parallel walls, rarely septate and branched. At immunohistochemistry, the hyphae were immunostained with polyclonal anti-P. insidiosum antibody in fragments of the cerebellum and nasal cavity. The findings showed that P. insidiosum rhinitis can secondarily affect the nervous system of sheep, causing nonspecific neurological clinical signs. [ABSTRACT FROM AUTHOR]

We aimed to genotype the South American clinical isolates of Pythium insidiosum using the single nucleotide polymorphisms (SNP) of the ribosomal DNA sequences (rDNA). Previously, an SNP-based multiplex-PCR was able to distinguish three different clades of P. insidiosum isolates. Thus, we used this assay to evaluate South American clinical isolates of P. insidiosum (n=32), standard strains from Costa Rica (n=4), Thailand (n=3), Japan (n=1), and India (n=1), a standard strain of Pythium aphanidermatum, and Brazilian environmental isolates of Pythium torulosum, Pythium rhizo-oryzae and Pythium pachycaule voucher (n=3). It was possible to allocate each American P. insidiosum isolate to clade I, the isolates of India, Japan, and Thailand to clade II, and the Thai isolate to clade III. P. aphanidermatum, P.torulosum, P.rhizo-oryzae and P.pachycaule voucher isolates were not amplified. For the first time, a P. insidiosum isolate from Uruguay, South America, was included in molecular analyzes. By SNP-based multiplex-PCR, it was possible to perform the identification and genotyping of the South American isolates of P. insidiosum, demonstrating similar genetic characteristics of these isolates. [ABSTRACT FROM AUTHOR]

Pythium insidiosum é um oomiceto aquático, responsável pela etiologia da pitiose, uma enfermidade crônica, observada freqüentemente em eqüinos. A produção de zoósporos móveis por este microrganismo se constitui no fator determinante da ocorrência da enfermidade. Este estudo avaliou a zoosporogênese e quantificou a produção de zoósporos de 32 amostras de Pythium insidiosum isoladas de eqüinos naturalmente infectados. Pythium insidiosum foi cultivado em meio Corn Meal Agar acrescido de fragmentos de grama, durante 5 dias, a 37°C. Posteriormente, os fragmentos de grama parasitados foram incubados em Meio de Indução a 37°C, por 24 horas. Observou-se que 16 amostras (50%) produziram 20.000 zoósporos mL-1, 12 isolados (37,5%) produziram acima de 20.000 zoósporos mL-1, enquanto quatro amostras (12,5%) produziram menos de 20.000 zoósporos mL-1. O período de maior produção de zoósporos foi entre 6 e 8 horas de incubação. O protocolo utilizado na indução da zoosporogênese mostrou-se eficiente e representa uma importante ferramenta, tanto para a identificação do Pythium insidiosum, como para a obtenção de zoósporos em quantidades suficientes para a inoculação em animais experimentais e aplicação no desenvolvimento de testes de suscetibilidade.Pythium insidiosum is an aquatic oomycete and the etiology of a chronic disease called pythiosis, commonly found in the skin of horses. The production of mobile zoospores by this microorganism is the determinant factor of this disease. This study evaluated the zoosporogenesis and quantification of zoospores in 32 samples of Pythium insidiosum isolated from horses with pythiosis. The assay used culture of the Pythium insidiosum in Corn Meal Agar plus grass blades for 5 days at 37°C. The grass blades were incubated in Induction Medium at 37°C for 24 hours. The findings showed 16 samples (50%) yielded 20,000 zoospores mL-1, 12 (37.5%) samples yielded over 20,000 zoospores mL-1 and 4 samples (12.5%) yielded less than 20,000 zoospores mL-1. The zoospores production was higher between 6 and 8 hours of incubation. The protocol used in the zoosporogenesis induction was efficient and represents an important tool for Pythium insidiosum identification and the attainment of zoospores in adequate amounts for inoculation in experimental animals and application in the development of susceptibility tests.

Cutaneous pythiosis is described in four horses in Northern Paraná, Brazil. All animals presented ulcerative, granulomatous, cutaneous lesions that did not invade adjacent muscular tissues. Histopathological evaluations revealed eosinophilic granulomatous reactions associated with intralesional hyphae suggestive of Pythium insidiosum observed at the margins of coagula. P. insidiosum hyphae were visualized by Gomori’s methenamine silver stain.