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.

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.

In vitro and ex vivo anti-Pythium insidiosum potential of ozonated sunflower oil.

This study sought to evaluate the in vitro and ex vivo susceptibility of Pythium insidiosum to ozonized sunflower oil (OSO) and verify the morphological alterations of OSO-exposed hyphae. Susceptibility assays were performed according to the broth microdilution protocol M38-A2/CLSI, and the minimal inhibitory (MIC) and minimal oomicidal (MOC) concentrations were also determined. Non-ozonated sunflower oil (SO) was used as the oil control. Additionally, kunkers from equine pythiosis were exposed to OSO. Damages caused by OSO and SO on P. insidiosum hyphae ultrastructure were verified using scanning electron microscopy. The MIC range for OSO was 7000 to 437.5 mg/mL, and the values for SO were higher, ranging from 56000 to 14000 mg/mL. The MOC was equal to MIC for both oil formulations. The OSO fully inhibited the oomycete growth from kunkers, although there was P. insidiosum growth in the kunker control in 24 h of incubation. The SEM analyses showed that both OSO and SO caused morphological alterations in P. insidiosum hyphae, highlighting the presence of cavitation along the hyphae with loss of continuity of the cell wall, which was more evident in the OSO-treated hyphae. The OSO had the best oomicidal activity, leading us to believe that our findings may support future research containing this formulation to be applied in integrative medicine protocols to control pythiosis in animals and humans.

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.

Exposure of Culex quinquefasciatus to the oomycete Pythium insidiosum: A protocol for in vitro studies.

Pythium insidiosum causes pythiosis, an infection that affects different species of mammals, including humans, and inhabits marshy ecosystems of tropical, subtropical, and temperate regions worldwide. Therefore, this study proposes a protocol to expose Culex quinquefasciatus to P. insidiosum zoospores. Cx. quinquefasciatus immatures (eggs, larvae, and pupae) were exposed to zoospores (8x103 zoospores/mL) of the oomycete for 24 h. The exposure of Cx. quinquefasciatus to the zoospores from L1 to the emergence of adults was evaluated, and P. insidiosum detection was performed by microbiological culture, polymerase chain reaction, and histopathological analysis of stage 4 larvae. The protocol used to produce Cx. quinquefasciatus colonies and adapted for this study proved viable for research on the interaction between P. insidiosum and this Culicidae species. Moreover, P. insidiosum presence was evident in all larval stages of the mosquito, although the presence of the oomycete was not detected in the eggs, pupae, and adults. This study is a pioneer in the development of a protocol to evaluate Cx. quinquefasciatus exposure to P. insidiosum zoospores, and under experimental conditions, P. insidiosum can establish itself in Cx. quinquefasciatus larval stages. The developed protocol is expected to serve as a basis for developing studies to evaluate the interactions of P. insidiosum with these mosquitoes and shed more light on the participation of culicids in expanding the ecological niche of P. insidiosum.

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.

Anti-Pythium insidiosum activity of bioactive compounds from medicinal plants.

Pythiosis is a serious disease caused by the aquatic oomycete Pythium insidiosum that mainly affects mammals. Unlike fungal and bacterial resistance induced by the indiscriminate use of drugs, P. insidiosum has low susceptibility to antifungal drugs. In this sense, essential oils and their major components emerge as a promising treatment line for this disease. Given the above, this study sought to verify P. insidiosum (n = 34) susceptibility to the bioactive compounds eugenol, α-terpineol, menthol, and carvacrol and correlate them with the respective essential oils of Eugenia caryophyllata, Melaleuca alternifolia, Mentha piperita, and Origanum vulgare. The essential oils and bioactive compounds were purchased commercially and tested according to the Clinical and Laboratory Standards Institute protocol M38-A2. Our findings showed that eugenol, α-terpineol, and carvacrol had superior anti-P. insidiosum action than their respective essential oils, suggesting that they may be responsible for inhibitory activity against P. insidiosum. Notably, the major compound with the best anti-P. insidiosum activity was α-terpineol; nonetheless, menthol showed less activity than its essential oil. The results imply that essential oils and their major compounds may be important allies in treating pythiosis, expanding the perspectives of developing new drugs with anti-P. insidiosum activity.

Identification and Characterization of Pythium, Globisporangium, and Phytopythium Species Present in Floricultural Crops from Long Island, New York.

Several Pythium, Globisporangium, and Phytopythium species cause Pythium diseases in greenhouse floricultural crops, resulting in significant seasonal losses. Four hundred and eighteen Pythium, Globisporangium, and Phytopythium isolates from flowering crops, growing media, or bench and floor debris were collected from Long Island greenhouses or clinic samples between 2002 and 2013. Isolates were identified to species based on morphology and internal transcribed spacer barcoding. Twenty-two species of Pythium, Phytopythium, and Globisporangium were identified, with Globisporangium irregulare sensu lato (s.l.) being the most common. To determine the origin of inoculum during the 2011 cropping season, 11 microsatellite loci were analyzed in 124 G. irregulare s.l. isolates collected in four greenhouses and six previously collected from clinic samples. Cluster analyses grouped G. irregulare s.l. isolates into four groups: G. irregulare sensu stricto, plus three G. cryptoirregulare clusters. The population structure defined by greenhouse and host was found in two clades. Additionally, the population dynamics of G. irregulare s.l. isolates associated with Pelargonium spp. from 2011 to 2013 were examined using 85 isolates and nine informative microsatellite loci to assess inoculum survival over multiple cropping seasons. Although most isolates had unique genotypes, closely related genotypes were found in the same locations over different years. Our results indicate that G. irregulare s.l. inocula have local as well as remote origins. Isolates may be initially brought into ornamental operations from common sources, such as infected plant materials or infested potting mixes. Our results support the hypothesis that established strains can serve as inocula and survive in greenhouse facilities over multiple seasons.

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.

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.

Experimental Pythium aphanidermatum Infection in Rabbits.

Pythium aphanidermatum is a common plant pathogen, reportedly capable of human infection. This study investigated whether P. aphanidermatum can infect domestic rabbits. Four rabbits were inoculated with P. aphanidermatum subcutaneously and evaluated for 30 days. Blood and tissue samples were collected from all animals. The animals developed palpable nodules 5-10 days after inoculation. Necropsy revealed caseous nodular lesions characterized histopathologically by pyogranulomatous inflammation containing hyphae in the necrotic areas. P. aphanidermatum was isolated from the caseous material and anti-P. aphanidermatum antibodies were detected by serological examination. This is the first report of experimental P. aphanidermatum infection in mammals. The results demonstrate that this oomycete can produce infection at the inoculation site and induce an immune response. The results suggest that P. aphanidermatum may be undergoing adaptation to vertebrate hosts.

Pythium and Phytopythium species associated with weeds collected in vegetable production fields in Brazil.

This study aimed to identify Pythium and Phytopythium species from weeds collected in vegetable fields and test their pathogenicity. Weeds with symptoms of damping-off, root rot or wilt were sampled in the Brazilian states of Ceará, Goiás and Pernambuco, as well as in the Distrito Federal, for isolation and identification of the causal agents. Once isolated, colonies with typical Pythium and Phytopythium characteristics grew in selective V8 medium. Procedures for species identification included morphology and amplification of the ITS and Cox II regions, which were compared with other accessions available at GenBank. The phylogenetic relationships among the isolates and pathogenicity to their original hosts were evaluated. Six Pythium species were identified: P. aphanidermatum, P. oopapillum, P. orthogonon, P. ultimum var. ultimum, P. myriotylum and P. sylvaticum, and two species of Phytopythium, Phy. chamaehyphon and Phy. oedochilum. In the pathogenicity tests, the 10 weed hosts showed symptoms of damping-off or root rot after inoculation, with exception of Portulaca oleraceae in which none of the isolates was pathogenic. Therefore, common weeds in vegetable fields areas can host different Pythium and Phytopythium species and play an important role in the epidemiology of vegetable diseases, in particular on pathogen survival and population increase.

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.

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.

Pitiose gástrica em canino jovem / Gastric pythiosis in a young canine

Pitiose gástrica canina é uma afecção crônica granulomatosa causada pelo pseudo-fungo Pythium insidiosum que ocasiona espessamento trasnsmural do estômago. Relata-se um caso de gastrite fúngica em um canino dobermann de 1 ano, macho, atendido no Hospital Veterinário da Universidade Federal do Mato Grosso, campus Sinop-MT, com sinais clínicos de diarreia escura intermitente, regurgitação, êmese e anorexia progressiva por cerca de três meses. No exame ultrassonográfico, observou espessamento gástrico difuso. Devido ao prognóstico ruim, foi realizado eutanásia. Na macroscopia, havia espessamento difuso da parede do estômago e petéquias multifocais por toda a mucosa. Na microscopia havia acentuado infiltrado inflamatório granulomatoso por toda a submucosa e muscular. Foi realizado coloração de metenamina nitrato de prata de Grocott (GMS), com evidenciação de hifas cenocíticas, raras septação e diâmetro de 6-10µm. A confirmação do diagnóstico e identificação do agente foi realizado através Imuno-histoquímica com anticorpo primário para Pythium insidiosum.

Canine gastric pythiosis is a chronic granulomatous infection caused by the insidious pythi fungus that causes stomach thickening. We report a case of fungal gastritis in a 1-year-old male progressive Doberman canine treated at the Veterinary Hospital of the Federal University of Mato Grosso, Sinop-MT campus, with clinical signs of intermittent diarrhea, regurgitation, vomiting and anorexia for about three months. On ultrasound examination, diffuse gastric thickening was observed. Due to the poor prognosis, euthanasia was performed. Macroscopically, there was diffuse thickening of the stomach wall and multifocal petechiae throughout the mucosa. On microscopy, there was a marked granulomatous inflammatory infiltrate throughout the submucosa and muscle Production of Grocott silver nitrate methenamine (GMS) was performed with evidence of cenocytic hyphae, rare septation and diameter of 6-10µm. Confirmation of the diagnosis and identification of the agent was performed through Immunohistochemistry with primary antibody to Pythium insidiosum.

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.