Equidae pythiosis in Brazil and the world: a systematic review of the last 63 years (1960-2023).

This systematic review compiles reports of clinical pythiosis in horses, mules and donkeys from 1960 to 2023 worldwide, focusing on Brazil. We searched databases and included 71 articles detailing clinical characteristics, geographic distribution, epidemiology, diagnostic methods, therapies, and outcomes. The results showed that publications on equine pythiosis have significantly increased since 2010. Brazil reported the highest incidence, comprising 55% of cases, predominantly in the southern, northeastern, and central-western regions during summer and autumn. Cutaneous pythiosis was the most prevalent form, generally presenting as single lesions in the appendicular region, and affected females more than males. Diagnosis typically involved histopathology, used alone or with other methods. Various treatments have been employed, with surgery, often combined with chemotherapy and immunotherapy, being the most common. Notably, 80.84% of treated animals recovered, highlighting the effectiveness of these therapies in enhancing survival rates. The limitations of the study included the lack of data in published case reports, which made it difficult to collect and calculate epidemiological data. Additionally, we recognize that pythiosis in Brazil is underreported, since this disease does not have mandatory notification and several cases are not registered and/or reported in the literature. Lastly, it is hypothesized that equid pythiosis may be more widespread than currently known, and its real occurrence in Brazil remains uncertain.

Treating Pythiosis with Antibacterial Drugs Targeting Protein Synthesis: An Overview.

This review article explores the effectiveness of antibacterial drugs that inhibit protein synthesis in treating pythiosis, a difficult-to-treat infection caused by Pythium insidiosum. The article highlights the susceptibility of P. insidiosum to antibacterial drugs, such as macrolides, oxazolidinones, and tetracyclines. We examine various studies, including in vitro tests, experimental infection models, and clinical case reports. Based on our synthesis of these findings, we highlight the potential of these drugs in managing pythiosis, primarily when combined with surgical interventions. The review emphasizes the need for personalized treatment strategies and further research to establish standardized testing protocols and optimize therapeutic approaches.

First confirmed case of equine pythiosis in Northern Veracruz, Mexico.

A clinical case of an adult horse with invasive, ulcerative, proliferative, pyogranulomatous disease of the skin (tumor) in the shoulder region is presented. The mass had a granulomatous and crater-shaped appearance, with serosanguinous discharge and the presence of fistulas with caseous material. The tumor was removed by surgery and sent to the laboratory for diagnosis. Histopathology was performed using Grocott-Gomori methenamine silver stain. The presence of necrotic material, fibrosis, infiltrated cells, and brown-colored hyphae, characteristic of members of the genus Pythium, were observed. To identify the infecting species, conventional PCRs for the amplification of the ITS-1 was carried out. Histopathological and PCR tests confirmed infection by a Pythium insidiosum strain closely associated with previous records from the US and Central America. Our report represents the first molecularly confirmed case of equine pythiosis in Mexico.

Pythium insidiosum: insights into biofilm formation and antibiofilm activity of antifungal drugs.

In this study, we investigate the ability of Pythium insidiosum to form biofilms across various substrates and the antibiofilm efficacy of 8-hydroxyquinoline derivatives (8-HQs). Biofilms of P. insidiosum were cultured on polystyrene plates, contact lenses, and horsehair. We provide the first evidence of P. insidiosum's biofilm-forming capability, thus considerably expanding our understanding of its transmission and pathogenesis. Our results demonstrate that 8-HQs effectively inhibit biofilm formation and eradicate pre-existing biofilms, underscoring their potential as a novel treatment strategy for pythiosis, a disease currently lacking a gold-standard treatment. This finding has particular relevance for ocular pythiosis associated with contact lens usage and potential infection sources in animals. Our results contribute to the scientific knowledge base and directly impact innovative therapeutic interventions' development.

Oomicidal activity of polypyrrole nanoparticles against Pythium insidiosum.

This study evaluated in-vitro action of a new molecule, the polypyrrole nanoparticles (Ppy-NP), against Pythium insidiosum isolates using M38-A2/CLSI; the minimal inhibitory (MIC) and minimal oomicidal (MOC) concentrations were also determined. Additionally, changes in the hyphae wall of P. insidiosum CBS 575.85 treated with Ppy-NP were evaluated by scanning electron microscopy (SEM). The MIC100 and MOC for all isolates ranged from 8 to 32 µg mL-1, and the MIC90 and MIC50 were 16 µg mL-1. The SEM showed structural damage to the hyphae of P. insidisoum treated with Ppy-NP, as hyphae surfaces with less turgidity were found, thereby showing scaling and ruptures compared to the control (untreated hyphae). Our findings highlighted the anti-P. insidiosum properties of Ppy-NP proved to be a promising candidate for research using pythiosis experimental models.

Cutaneous pythiosis in equines in the Amazon Biome / Pitiose cutânea em equídeos no Bioma Amazônico

The study aimed to describe the clinicopathological aspects of 37 cases of pythiosis, 34 in horses and three in mules, from properties located in the Amazon biome of Pará, Brazil. The clinical signs observed in the animals were weakness, poor-to-regular nutritional status, pale mucous membranes, itching at the lesion site, and lameness when the limbs were affected. The lesions were located on the lips, nostrils, rib region, thoracic and abdominal walls, scapular, distal limbs, foreskin, perineum and udder. Macroscopically, ulcerative and granulation-tissue-like masses were observed, with fistulous tracts filled with yellowish and foul-smelling serosanguinous discharges. In the biopsy, it was possible to visualize white and firm areas with foci of yellowish necrotic material and hardened masses, called kunkers. The histopathological examination showed a pyogranulomatous inflammatory reaction with the presence of Pythium insidiosum hyphae, which were impregnated with black, confirming the diagnosis of pythiosis in equids in the Amazon biome, being the first report of the disease in mules in the region.

O trabalho objetivou descrever os aspectos clínico-patológicos de 37 casos suspeitos de pitiose, 34 em equinos e três em muares, provenientes de propriedades localizadas no Pará, bioma amazônico brasileiro. Os sinais clínicos observados nos animais eram caracterizados por debilidade, estado nutricional de ruim a regular, mucosas pálidas, prurido no local da lesão, além de claudicação quando os membros foram acometidos. As lesões eram localizadas nos lábios, narinas, região das costelas, parede torácica e abdominal, escapular, distais dos membros, prepúcio, períneo e úbere. Macroscopicamente observavam-se extensas lesões ulceradas com intensa proliferação de tecido de granulação, de bordos irregulares, com tratos fistulosos, de consistência firme, denominados de "kunkers", preenchidos com material amarelado e friável, possuindo exsudação serossanguinolenta de odor fétido. Ao exame histopatológico observou-se reação inflamatória piogranulomatosa com presença de hifas de Pythium insidiosum, as quais se impregnaram de negro, confirmando o diagnóstico de pitiose em equídeos no Bioma Amazônico, sendo o primeiro relato da doença em muares na região.

Promising use of nanotechnology in Pythium insidiosum: a systematic review / Utilização promissora de nanotecnologia em Pythium insidiosum: uma revisão sistemática

ABSTRACT: 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.

RESUMO: O oomiceto aquático Pythium insidiosum é um patógeno emergente de relevância em medicina humana e veterinária. É o agente etiológico da pitiose, uma enfermidade de distribuição mundial, que acomete principalmente em equinos, caninos e seres humanos, podendo apresentar-se nas formas cutâneas, subcutâneas, oculares, gastrointestinais e sistêmicas. Considerando que os métodos terapêuticos disponíveis para o tratamento da doença não são completamente efetivos, há uma necessidade de investigar formas de tratamentos com melhor eficácia, como os compostos de diferentes classes farmacológicas, compostos de origem natural, bem como, novas alternativas tecnológicas, incluindo a nanotecnologia. Deste modo, este trabalho objetivou avaliar publicações científicas referentes a utilização de nanotecnologia em P. insidiosum. Para isso, realizou-se uma revisão sistemática da literatura, buscando artigos no período de 2010 a 2022, nas bases de dados LILACS, MEDLINE, Google Scholar, PubMed e SciELO, utilizando-se os descritores Pythium insidiosum, pitiose, nanotecnologia, nanopartículas, nanoemulsão e tratamento. Encontrou-se 162 artigos com familiaridade a temática pesquisada; no entanto, apenas quatro estudos foram incluídos, pois atendiam os critérios estabelecidos na pesquisa. Para análise estatística dos dados obtidos nos estudos in vitro, utilizou-se meta-análise. Demonstrou-se o promissor uso de nanotecnologia como alternativa no desenvolvimento de compostos antimicrobianos com atividade anti-P. insidiosum. Entretanto, constata-se que estudos adicionais se fazem necessários para verificar o potencial uso desta tecnologia na terapêutica clínica contra infecções por P. insidiosum.

Meningoencephalitis secondary to rhinitis caused by Pythium insidiosum in sheep / Meningoencefalite secundária a rinite por Pythium insidiosum em um ovino

ABSTRACT: 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.

RESUMO: Relata-se um caso de meningoencefalite por Pythium insidiosum secundária a rinite em uma ovelha mestiça, três anos de idade, proveniente de um rebanho de 15 animais, criados extensivamente e com acesso livre a açude. O animal apresentava dispneia leve, cegueira, midríase, opistótono, nistagmo, incoordenação, diminuição do tônus mandibular e espasticidade dos membros pélvicos. O exame macroscópico da cavidade nasal evidenciou uma massa vermelho-escura, irregular, friável, que comprometia bilateralmente o septo nasal e a porção rostral dos cornetos nasais. No encéfalo, havia espessamento difuso das leptomeninges do cerebelo e porção ventral do tronco encefálico caracterizado por material granular amarelado associado à hiperemia dos vasos. No assoalho do quarto ventrículo, havia deposição de material amarelado, irregular, levemente granuloso, que se projetava em direção ao óbex e deslocava o cerebelo dorsolateralmente. Microscopicamente, havia rinite piogranulomatosa, eosinofílica, necrosante e meningoencefalite fibrinossupurativa, eosinofílica, necrosante, ambas associadas à trombose, vasculite e hifas intralesionais. Pela utilização da técnica de GMS as hifas foram impregnadas pela prata e apresentavam paredes finas e paralelas, raramente septadas e ramificadas. Na imuno-histoquímica, houve imunomarcação com o anticorpo policlonal anti-P. insidiosum em fragmentos do cerebelo e cavidade nasal. Os achados evidenciaram que rinite por P. insidiosum pode afetar secundariamente o sistema nervoso de ovinos, causando sinais clínicos neurológicos inespecíficos.

Meningoencephalitis secondary to rhinitis caused by Pythium insidiosum in sheep / Meningoencefalite secundária a rinite por Pythium insidiosum em um ovino

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.

Relata-se um caso de meningoencefalite por Pythium insidiosum secundária a rinite em uma ovelha mestiça, três anos de idade, proveniente de um rebanho de 15 animais, criados extensivamente e com acesso livre a açude. O animal apresentava dispneia leve, cegueira, midríase, opistótono, nistagmo, incoordenação, diminuição do tônus mandibular e espasticidade dos membros pélvicos. O exame macroscópico da cavidade nasal evidenciou uma massa vermelho-escura, irregular, friável, que comprometia bilateralmente o septo nasal e a porção rostral dos cornetos nasais. No encéfalo, havia espessamento difuso das leptomeninges do cerebelo e porção ventral do tronco encefálico caracterizado por material granular amarelado associado à hiperemia dos vasos. No assoalho do quarto ventrículo, havia deposição de material amarelado, irregular, levemente granuloso, que se projetava em direção ao óbex e deslocava o cerebelo dorsolateralmente. Microscopicamente, havia rinite piogranulomatosa, eosinofílica, necrosante e meningoencefalite fibrinossupurativa, eosinofílica, necrosante, ambas associadas à trombose, vasculite e hifas intralesionais. Pela utilização da técnica de GMS as hifas foram impregnadas pela prata e apresentavam paredes finas e paralelas, raramente septadas e ramificadas. Na imuno-histoquímica, houve imunomarcação com o anticorpo policlonal anti-P. insidiosum em fragmentos do cerebelo e cavidade nasal. Os achados evidenciaram que rinite por P. insidiosum pode afetar secundariamente o sistema nervoso de ovinos, causando sinais clínicos neurológicos inespecíficos.

Mefenoxam and pyraclostrobin: toxicity and in vitro inhibitory activity against Pythium insidiosum.

The objective of this study is to verify in vitro susceptibility of Pythium insidiosum against the agricultural fungicides mefenoxam and pyraclostrobin and evaluate the toxicity of both compounds. Twenty-one P. insidiosum isolates were tested against mefenoxam and pyraclostrobin using the broth microdilution method. Minimum inhibitory and oomicidal concentrations for both compounds were established. Additionally, scanning electron microscopy was performed on P. insidiosum hyphae treated with the sublethal concentration of each fungicide. The toxicity of the compounds was evaluated in vivo Caenorhabditis elegans model. The concentration to inhibit 100% of P. insidiosum growth ranged from 0·625 to 10 µg ml-1 for mefenoxam and from 0·019 to 5 µg ml-1 for pyraclostrobin. The SEM analysis revealed changes on the surface of the hyphae treated with the fungicides, suggesting possible damage caused by these compounds. There was no evidence of toxicity in vivo models. Mefenoxam and pyraclostrobin did not show toxicity at the doses evaluated and have inhibitory effects on the pathogenic oomycete P. insidiosum. However, further evaluations of their pharmacokinetics and toxicity in different animal species and possible pharmacological interactions are necessary to infer a possible use in the clinical management of pythiosis.

A Retrospective Study of Pythiosis in Domestic Animals in Northeastern Brazil.

Pythiosis is an endemic disease in northeastern Brazil and we now report the epidemiological, clinical and pathological findings in a retrospective study of naturally occurring cases in domestic animals. From January 1985 to December 2020, the Laboratory of Animal Pathology of the Federal University of Campina Grande examined 13,542 tissue samples from necropsies and biopsies. Among these samples, 306 were diagnosed as pythiosis: 195 cases in horses, 75 in sheep, 19 in dogs, six in mules, four in cattle, three in cats, two in goats, one in a donkey and one in an ostrich. Affected equids had lesions in the skin, mammary glands and nasal cavities. Affected sheep had cutaneous, nasal and digestive lesions while cattle and goats had cutaneous lesions. Carnivores developed lesions, mainly in the alimentary tract, of sufficient severity to cause death or result in euthanasia. The single affected bird had lesions in the alimentary tract and surgical excision resulted in remission. The disease had a long and life-threatening clinical course in most affected species but resolved spontaneously in cattle. Clinical signs were directly related to the location of the lesions, which were invariably characterized by chronic inflammation associated with intralesional hyphae. Veterinary clinicians and pathologists should be familiar with the clinicopathological features of pythiosis and the wide range of susceptible animal species.

Pythium insidiosum complex hides a cryptic novel species: Pythium periculosum.

Early phylogenetic analysis of Pythium insidiosum, the etiologic agent of pythiosis in mammals, showed the presence of a complex comprising three monophyletic clusters. Two included isolates recovered from cases of pythiosis in the Americas (Cluster I) and Asia (Cluster II), whereas the third cluster included four diverged isolates three from humans in Thailand and the USA, and one isolate from a USA spectacled bear (Cluster III). Thereafter, several phylogenetic analyses confirmed the presence of at least three monophyletic clusters, with most isolates placed in clusters I and II. Recent phylogenetic analyses using isolates from environmental sources and from human cases in India, Spain, Thailand, and dogs in the USA, however, showed the presence of two monophyletic groups each holding two sub-clusters. These studies revealed that P. insidiosum possesses different phylogenetic patterns to that described by early investigators. In this study, phylogenetic, population genetic and protein MALDI-TOF analyses of the P. insidiosum isolates in our culture collection, as well as those available in the database, showed members in the proposed cluster III and IV are phylogenetically different from that in clusters I and II. Our analyses of the complex showed a novel group holding two sub-clusters the USA (Cluster III) and the other from different world regions (Cluster IV). The data showed the original P. insidiosum cluster III is a cryptic novel species, now identified as P. periculosum. The finding of a novel species within P. insidiosum complex has direct implications in the epidemiology, diagnosis, and management of pythiosis in mammalian hosts.

Melaleuca alternifolia formulations in the treatment of experimental pythiosis.

Essential oils (EO) are aromatic compounds from the plant secondary metabolism. Melaleuca alternifolia EO is well known for its medicinal properties and promising use as an antimicrobial agent. Pythiosis is a difficult-to-treat and emerging disease caused by the oomycete Pythium insidiosum. This study evaluated a nanoemulsion formulation of M. alternifolia (NEMA) in topical and intralesional application to treat experimental pythiosis. Dermal toxicity tests were performed on M. alternifolia EO in Wistar rats. Pythiosis was reproduced in rabbits (n = 9) that were divided into groups: group 1 (control), cutaneous lesions with daily topical application of a non-ionizable gel-based formulation and intralesional application of sterile distilled water every 48 h; group 2 (topical formulation), lesions treated daily with topical application of a non-ionizable gel-based formulation containing 5 mg/ml of NEMA; and group 3 (intralesional formulation), lesions treated with NEMA at 5 mg/ml in aqueous solution applied intralesionally/48 h. The animals were treated for 45 days, and the subcutaneous lesion areas were measured every 5 days. M. alternifolia EO showed no dermal toxicity. The lesion areas treated with intralesional NEMA reduced at the end of treatment, differing from groups 1 and 2 (P < 0.05). In the topically treated group, the lesion areas did not differ from the control group, although the number of hyphae significantly reduced (P < 0.05). Under the experimental conditions of this study, the NEMA formulations presented a favorable safety profile. However, further studies are required to evaluate if this safety applies to higher concentrations of NEMA and to validate its use in clinical pythiosis.

Canine gastrointestinal pythiosis: a case report in Brazilian Pantanal from a diagnostic approach / Pitiose gastrointestinal canina: relato de caso no Pantanal brasileiro a partir de uma abordagem diagnóstica

Pythiosis is caused by an aquatic fungus-like organism (Pythium insidiosum). It is considered an important public health issue as it can affect both animals and humans. This paper reports a case of gastrointestinal pythiosis in a dog. The patient was hospitalized for four days, during which the animal received supportive and symptomatic treatment. But the applied treatment was unsuccessful and the dog's clinical condition worsened, culminating in death. Complementary imaging tests such as radiography and ultrasonography, as well as hematological tests, were performed during the hospitalization period. The definitive diagnosis was reached in the postmortem as macroscopic and microscopic characteristics suggested the presence of intestinal granuloma and accentuated multifocal suppurative necrotic enteritis. Additionally, the histological evaluation revealed morphological structures compatible with P. insidiosum. Also, the results of nested PCR performed showed partial amplification (105 bp) of the ITS1 region of the ribosomal gene of P. insidiosum.(AU)

A pitiose é causada por um organismo aquático semelhante a um fungo (Pythium insidiosum) e considerada um importante problema de saúde pública, pois pode afetar animais e humanos. Este artigo relata um caso de pitiose gastrointestinal em um cão. O paciente ficou internado por quatro dias, período em que o animal recebeu tratamento de suporte e sintomático. No entanto, o tratamento aplicado não teve sucesso e o quadro clínico do cão piorou, culminando com a morte. Exames de imagem complementares, como radiografia e ultrassonografia, bem como exames hematológicos, foram realizados durante o período de internação. O diagnóstico definitivo foi feito na autópsia, pois as características macroscópicas e microscópicas sugeriam a presença de granuloma intestinal e acentuada enterite necrótica multifocal supurativa. Além disso, a avaliação histológica revelou estruturas morfológicas compatíveis com P. insidiosum. Além disso, a nested PCR foi realizada e mostrou amplificação parcial (105 pb) da região ITS1 do gene ribossomal de P. insidiosum.(AU)

Anti-Pythium insidiosum activity of MSI-78, LL-37, and magainin-2 antimicrobial peptides.

We investigated the anti-Pythium insidiosum activity of the antimicrobial peptides (AMPs) MSI-78, LL-37, and magainin-2. To detect the minimum inhibitory concentration (MIC), fourteen clinical strains were incubated with the AMPs following the CLSI M38-A2 protocol. All three AMPs showed antimicrobial activity with an MIC range of 20-80 mg/L against all strains. We concluded that the evaluated AMPs have great potential as anti-Pythium insidiosum agents, and their activity deserves to be more explored in further research. Antimicrobial peptides were tested against Pythium insidiosum, a microorganism that causes a difficult-to-treat disease in animals and humans. These peptides have been shown to be able to kill P. insidiosum and may be candidates for use in the treatment of this infection.

Clioquinol and 8-hydroxyquinoline-5-sulfonamide derivatives damage the cell wall of Pythium insidiosum.

AIMS: To evaluate the antimicrobial activity and to determine the pharmacodynamic characteristics of three 8-hydroxyquinoline derivatives (8-HQs) against Pythium insidiosum, the causative agent of pythiosis. METHODS AND RESULTS: Antimicrobial activity was tested by broth microdilution and MTT assays. The antimicrobial mode of action was investigated using sorbitol protection assay, ergosterol binding assay, and scanning electron microscopy. Clioquinol, PH151, and PH153 were active against all isolates, with MIC values ranging from 0.25 to 2 µg ml-1. They also showed a time- and dose-dependent antimicrobial effect, damaging the P. insidiosum cell wall. CONCLUSIONS: Together, these results reinforce the potential of 8-HQs for developing new drugs to treat pythiosis.

Clioquinol and 8-hydroxyquinoline-5-sulfonamide derivatives damage the cell wall of Pythium insidiosum.

AIMS: To evaluate the antimicrobial activity and to determine the pharmacodynamic characteristics of three 8-hydroxyquinoline derivatives (8-HQs) against Pythium insidiosum, the causative agent of pythiosis. METHODS AND RESULTS: Antimicrobial activity was tested by broth microdilution and MTT assays. The antimicrobial mode of action was investigated using sorbitol protection assay, ergosterol binding assay, and scanning electron microscopy. Clioquinol, PH151, and PH153 were active against all isolates, with MIC values ranging from 0.25 to 2 µg ml-1. They also showed a time- and dose-dependent antimicrobial effect, damaging the P. insidiosum cell wall. CONCLUSIONS: Together, these results reinforce the potential of 8-HQs for developing new drugs to treat pythiosis.

Outbreak of equine pythiosis in a southeastern region of Brazil: Environmental isolation and phylogeny.

Pythiosis is a disease caused by the oomycete Pythium insidiosum, mainly reported in equines, dogs and humans and directly transmitted through contaminant zoospores in aquatic environments. We report the first outbreak of equine pythiosis in five equines. Wound samples were submitted for diagnostic testing including mycological culture and nested PCR. Treatment approaches consisted of conventional and alternative therapies. Microbiological analyses were performed using water samples from the riverbanks close to where the animals had grazed. All animals were positive for P. insidiosum cultures, and two animals responded successfully to alternative therapy (ozone therapy). After culture and molecular analysis of environmental samples, the presence of P. insidiosum in one section of the Tietê River was confirmed through a 99% sequence identity. Phylogenetic analyses using the cytochrome oxidase II gene showed that the animal isolates clustered in clade I and the environmental isolates clustered in clade III. Although the environmental and wound isolates belonged to different genetic clades, we concluded that the Tietê River is an important source of infection by P. insidiosum and that research concerning environmental isolation of P. insidiosum from rivers and lakes should be strongly facilitated in Brazil.

Pythiosis in Equidae in Northeastern Brazil: 1985-2020.

The epidemiologic, clinical, pathologic, microbiological and immunohistochemical findings of pythiosis in equidae in northeastern Brazil are described. From January 1985 to December 2020 the Laboratory of Animal Pathology of the Federal University of Campina Grande received 1,331 tissue samples of equidae, 202 (15.17%) of which were diagnosed as pythiosis. Equidae of both sexes with ages varying from 4 months to 25 years were affected. Most animals were mixed breed (79.7%) and reared in an extensive system (73.26%). The disease occurred throughout the year but the highest incidence (70.29%) was noted after the rainy season. The clinical course was always chronic. The lesions were preferentially located on the limbs and ventral thoracoabdominal wall and characterized by nodules or tumor-like masses with ulcerations and serosanguineous discharge. The cut surface showed fistulous tracts containing kunkers. The direct examination of the kunkers and microbiological culture revealed sparsely septate and branched hyaline hyphae. Histopathology revealed a marked inflammatory infiltrate of eosinophils with multifocal well-defined areas of eosinophil necrosis and collagenolysis and intralesional negatively-stained hyphal profiles; in the donkey, a pyogranulomatous inflammatory infiltrate was noted surrounding these areas. Immunohistochemistry for Pythium insidiosum revealed strong immunolabelling of the hyphae. Pythiosis occurs endemically in equidae in northeastern Brazil, with seasonal variation in the incidence. The intralesional kunkers establishes an accurate presumptive diagnosis, but confirmation should preferably be performed through histopathology associated with immunohistochemistry, culture-based or molecular methods.

Prospecting Biomarkers for Diagnostic and Therapeutic Approaches in Pythiosis.

Pythiosis, whose etiological agent is the oomycete Pythium insidiosum, is a life-threatening disease that occurs mainly in tropical and subtropical countries, affecting several animal species. It is frequently found in horses in Brazil and humans in Thailand. The disease is difficult to diagnose because the pathogen's hyphae are often misdiagnosed as mucoromycete fungi in histological sections. Additionally, there is no specific antigen to use for rapid diagnosis, the availability of which could improve the prognosis in different animal species. In this scenario, we investigated which P. insidiosum antigens are recognized by circulating antibodies in horses and humans with pythiosis from Brazil and Thailand, respectively, using 2D immunoblotting followed by mass spectrometry for the identification of antigens. We identified 23 protein spots, 14 recognized by pooled serum from horses and humans. Seven antigens were commonly recognized by both species, such as the heat-shock cognate 70 KDa protein, the heat-shock 70 KDa protein, glucan 1,3-beta-glucosidase, fructose-bisphosphate aldolase, serine/threonine-protein phosphatase, aconitate hydratase, and 14-3-3 protein epsilon. These results demonstrate that there are common antigens recognized by the immune responses of horses and humans, and these antigens may be studied as biomarkers for improving diagnosis and treatment.