pythiosis is a chronic granulomatous disease caused by the oomycete pythium insidiosum belonging to the kingdom stramenopila 
 . these opportunistic pathogens live in warm stagnant water and are most often reported in regions with tropical and subtropical environments . in brazil , the disease occurs predominantly in horses , however , outbreaks have been reported in sheep and cattle , . pythiosis has three distinct forms of clinical presentation in domestic animals : cutaneous , , , gastrointestinal   and rhinofacial . in cattle , the cutaneous form of the disease is sporadic and widely described   and usually occurs during the rainy season in subtropical areas . the clinical diagnosis is based on epidemiological characteristics and pathological lesions . however , for definitive diagnosis , cultivation and identification of the agent are crucial . techniques of immunohistochemistry , serological testing by elisa and polymerase chain reaction ( pcr ) are employed to establish the definitive diagnosis of the disease , . the aim of this study is to report the epidemiological , pathological and mycological findings of cutaneous pythiosis in cattle in southern brazil . during the month of february of 2016 , 23 calves presented ulcerated cutaneous lesions in thoracic and pelvic limbs , sometimes extending to the ventral thoracic region . the herd consisted of 150 beef cattle ( 100 adults and 50 calves ) in moderate body condition , belonging to a property in the city of triunfo in southern of brazil ( 295608.2 s 512754,2w ) . the cattle were kept next to a river , in native grass areas with extensive marshy regions ( fig . the cutaneous lesions were observed after an initial period of 15 days after the animals were introduced into the area and had a clinical course of 2030 days , with subsequent spontaneous regression in all affected calves after this period . calves affected received supportive therapy , to prevent secondary skin infections with basic wound management techniques . cutaneous biopsies were performed in eight calves , from which six samples were fixed in 10% neutral buffered formalin , processed routinely for histopathology , and stained with hematoxylin - eosin ( he ) and gomori methenamine silver ( gms ) . two samples were kept at room temperature , and later submitted to cultivation and isolation of the agent . grossly , the lesions were located predominantly in the distal regions of the thoracic and pelvic limbs , and sometimes in carpal - ulnar region of forelimbs and ventral thoracic region . these consisted of multifocal ulcerated areas , with approximately 5  cm in diameter , reddish surface , sometimes covered by crusts ( fig . histopathological examination revealed multifocal pyogranulomas in the superficial and deep dermis characterized by inflammatory infiltrate of neutrophils , eosinophils and a marked amount of epithelioid macrophages in the central region , surrounded by rare multinucleated giant cells ( fig . , there were still occasional transverse and longitudinal sections of non - stained hyphae , surrounded often by eosinophilic radiated material morphologically compatible with splendore - hoeppli reaction . skin sections stained with gms showed rare centrally located hyphae with approximately 49  m in diameter , ramified and rarely septate . these hyphae were also stained by immunohistochemistry , predominantly observed in the center of these pyogranulomas ( fig . 1d ) , employing the polyclonal antibody ( non - commercial ) anti - p . insidiosum produced in rabbits , at a 1:100 dilution in phosphate buffered saline ( pbs ) , developed with a red chromogen , 3-amino-9-ethylcarbazole ( aec , dako north america , carpinteria , usa ) . plates of polystyrene of 96 wells were sensitized with the antigen diluted in pbs and incubated overnight at 4  c . each well received 100  l of bovine serum albumin 0.2% and , after the incubation period , the plates were washed and stored at 4  c until the moment of the use . the tested serums were diluted at 1:2000 in pbs in ph 7.2 , distributed 100  l in each well in the plates and incubated at 37  c for 1  h. then , the plates were incubated with a specific secondary antibody for the species ( anti - igg bovine conjugated with peroxidase ) with dilution of 1:5000 . the plates then received the chromogen buffer ( ortho - phenylene - diamine ) and the reading was carried out through a spectrophotomer of plates with 490  nm . the determination of levels of antibodies by elisa showed positive results for the detection of anti - pythiumantibodies for all cattle , suggesting that these animals did develop pythiosis . the cutaneous biopsies were cultivated on corn meal agar ( cma ) at 37  c for 48  h. a hyaline colony with a short and whitish radiating mycelium was observed . the microscopic evaluation revealed the presence of sparsely septate hyphae similar to p. insidiosum growth . for molecular identification , total dna was extracted of the isolate according to the protocol described in the literature , . the sample was sequenced and data obtained were deposited in genbank under the accession number kx369614 . it is usually considered a sporadic disease , which occurs during the rainy season in subtropical areas . the diagnosis of cutaneous pythiosis in cattle in the present outbreak was obtained through the epidemiological and the pathological findings associated with the molecular techniques , serological analyses and cultivation of p. insidiosum . the epidemiological findings described ,   are similar to those seen in this study , in which only young animals that remained in swampy regions were affected . in a previous report , the time between introduction of the animals in flooded areas and the onset of clinical signs was 15 days with a clinical course that extended over a period of two months , similar to that observed in this study . high rainfall and heavy flooding in the region , influenced by el nio , suggests that the precipitation factor predisposed the occurrence of this outbreak associated with the presence of the animals for long periods in this marshy environment . the clinical and pathological findings observed in this study were similar to those described by other authors , , , , that reported ulcerated lesions with thickening of the dermis and edema in distal regions of fore and hind limbs . one aspect observed in this outbreak and not described to date , was the presence of lesions in the proximal regions located in radio - ulnar region of forelimbs and ventral thoracic region . the location of the lesions is therefore directly related to the parts of the body that stay in direct contact with water containing zoospores of p. insidiosum 
 . in bovines , histopathological lesions are characterized by multifocal pyogranulomas in superficial and deep dermis , composed of polymorphonuclear cells and a predominance of epithelioid macrophages and multinucleated giant cells . a limited number of ramified septate hyphae , surrounded by splendore - hoeppli reactions suggest deposition of antigen - antibody complexes around the agent , similarly observed in this study . pcr assays , immunohistochemistry , and immunoblot analyses have been developed for identification and differentiation of p. insidiosum 
 . the differentiation of p. insidiosum from other species and organisms that cause similar clinical signs is very important to select the appropriate treatment . pithiosis is a self - limiting condition in cattle , and spontaneous healing has been reported to occur after a few weeks to two months . thus , it does not require specific treatment , . in horses , it is a chronic disease and effective treatment is obtained with wide surgical excision followed by the use of immunotherapy . immunotherapic treatment in horses has an effectiveness of 72% when employed alone and of 90% when combined to surgical debridement of diseased tissue , , . the microbial culture is a traditional method to identify p. insidiosum ; however , it requires expertise to identify this specific microorganism . in this study the pcr test followed by sequencing confirmed the previous morphological identification in the cattle samples of the present work . the molecular techniques represent an important sensitive and specific diagnostic tool for pythiosis . the serological analysis by elisa , proved to be a specific , sensitive and rapid technical in the diagnosis of bovine pythiosis  . the combination of pathological , serological , cultivation and molecular techniques employed in this study were crucial and allowed to characterize the outbreak of cutaneous pythiosis in cattle in southern brazil . the author(s ) declared no potential conflicts of interest with respect to the research , authorship , and/or publication of this article .