Epidemiological Survey of Equine Pythiosis in the Brazilian Pantanal and Nearby Areas: Results of 76 Cases

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Journal of Equine Veterinary Science 34 (2014) 270–274

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Journal of Equine Veterinary Science

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Original Research Epidemiological Survey of Equine Pythiosis in the Brazilian Pantanal and Nearby Areas: Results of 76 Cases

Carlos E.P. dos Santos PhD a,*, Daniel G. Ubiali MSc b, Caroline A. Pescador PhD b, Régis A. Zanette MSc c, Janio M. Santurio PhD c, Luiz C. Marques PhD d

a Postgraduate Program in Veterinary Medicine, Sao Paulo State University, Jaboticabal, Brazil b Department of Veterinary Clinical Medicine, Laboratory of Veterinary Pathology, Faculty of Agronomy, Veterinary Medicine and Zootechny, Federal University of Mato Grosso, Cuiabá, Brazil c Laboratory of Mycological Research, Department of Microbiology, Federal University of Santa Maria, Santa Maria, Brazil d Faculty of Agrarian and Veterinary Sciences, Department of Veterinary Clinic and Surgery, UNESP, Jaboticabal, Brazil

article info abstract

Article history: A clinical epidemiological study was conducted among 34 rural properties located within Received 13 March 2013 the Brazilian Pantanal region and nearby areas between 2007 and 2010. The diagnosis of Received in revised form 7 May 2013 equine pythiosis was based on antibody detection (by enzyme-linked immunosorbent Accepted 13 June 2013 assay), polymerase chain reaction, histopathological analysis, and cultures positive for Available online 3 August 2013 Pythium insidiosum. The majority of the affected animals (85%) were in the Pantanal biome, which had a higher disease prevalence (0.9%-66.7%) than that of the Cerrado (2.7%-33.3%). Keywords: The disease was more prevalent in the rainy season (January-March), with an abrupt fall in Oomycete – 2 ¼ Pythium insidiosum the number of cases during the drought period (April September; correlation of R 0.77; < Epidemiology P .01). Generally, the average prevalence of equine pythiosis in both regions was 5%, with Equine mortality and lethality rates of 1.3% and 23.1%, respectively, in the Pantanal and 2.3% and 45.5%, respectively, in the Cerrado. However, the treatment with immunotherapy may have underestimated these numbers, especially in the Pantanal. Animals older than 1 year were 8.09 times more affected by the disease than younger animals in the same environment (P < .05). A correlation between the anatomical area of the lesion and the type of skin color was also observed. Approximately 73% of the lesions were found in dark- pigmented areas, and animals with a dark coat color were affected more frequently. These ﬁndings highlight the importance of hematophagous insects in the epidemiology of pythiosis because these areas are preferred for blood feeding. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction abundant serosanguineous discharge [1,3,4]. The P. insidiosum organism is essentially aquatic, which explains the Pythiosis is a life-threatening infectious disease in prevalence of the disease in people and animals that live in mammals and birds caused by the oomycete Pythium direct contact with stagnant water contaminated with insidiosum [1,2]. Horses are the animals most frequently zoospores, such as rice ﬁelds in Thailand and the Pantanal infected and typically develop ulcerative granulomas with biome in Brazil [5,6]. Small skin lesions, such as those caused by mosquito bites, are believed to be the locus of * Corresponding author at: Carlos E. P. dos Santos, PhD, Departamento penetration of P. insidiosum zoospores [1,7]. de Clínica Médica Veterinária, Faculdade de Agronomia, Medicina Vet- Equine pythiosis has been reported worldwide, although erinária e Zootecnia, Universidade Federal de Mato Grosso, Av. Fernando Correa da Costa 2367, Bairro Boa Esperança, Cuiabá, MT 78068-900, the Brazilian Pantanal probably contains the largest con- Brazil. centration of cases caused mainly by the combination of E-mail address: [email protected] (C.E.P. dos Santos). three factors: warm temperatures, ﬂooded lands, and high

0737-0806/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jevs.2013.06.003 C.E.P. dos Santos et al. / Journal of Equine Veterinary Science 34 (2014) 270–274 271 numbers of animals [4,6,8]. Treatment of the disease in serosanguineous discharge. Mean lesion diameters varied horses is based on radical surgical excision and/or immu- from approximately 7 cm in the first week to 15-20 cm notherapy. However, the rate of success is variable and within 3 weeks. Chronic lesions showed draining fistulas depends upon many factors [1,3], which has spurred the containing yellow-gray stony masses referred to as “kunk- research of different treatment options [9-12]. ers” [1] and were associated with weight loss in the animals. The disease poses significant economic losses for equine Definitive diagnosis was established by a positive result breeders in Brazil, but the numbers are unknown as are from at least two of the following procedures: indirect many aspects of its epidemiology and etiopathology. enzyme-linked immunosorbent assay (ELISA) [4], histo- Therefore, this study reports the epidemiological aspects of pathological examination of tissue specimens with Grocott 76 cases of P. insidiosum equine infection that occurred in silver stain, isolation of the pathogen and subsequent the Brazilian Pantanal and nearby areas. induction of zoosporogenesis [13], and polymerase chain reaction (PCR) assay of the lesions conducted with P. insid- 2. Materials and Methods iosum-specific primers [14].

2.1. Characterization of the Studied Area 2.4. Statistical Analysis

Data were obtained from 34 rural properties located Data were analyzed using Epi Info sofware version 6 within the subregion of Poconé, in the northern area of the (Centers for Disease Control and Prevention, Atlanta, GA) Pantanal Matogrossense, Brazil. Twenty-four properties [15]. The chi-square and Fisher exact tests were used to were located in the Pantanal biome, which is characterized evaluate the association among the studied variables. by the intercalation of clay and sandy soils commonly associated with swamps, and 10 properties were located in 3. Results and Discussion the Cerrado (Brazilian savannah) and ecotones, which are more permeable but seasonally waterlogged soils that Seventy-six cases of equine pythiosis were diagnosed in resemble the Pantanal. the studied area from 2007 to 2010. The triad of flooded Seasons in the studied regions are well defined, with the soil, abundance of aquatic vegetation, and high tempera- rainy period ranging from October to March and the tures are highly favorable for development and mainte- drought period ranging from April to September (average nance of the oomycete P. insidiosum [1,4,6,7,16]. This annual rainfall is between 800 and 1,200 mm). The hot and environment is characteristic of the Pantanal biome, where humid weather is characteristically tropical, with an 85.5% (65 of 76) of the cases were observed. The remaining average annual temperature of 25.5C and an average 14.5% (11 of 76) of the cases were observed in the Cerrado minimum temperature of 20C in the winter and 32Cin biome and in transitional areas between the Pantanal and the summer. the Cerrado. The presence of naturally flooded areas Climate data including the average temperature and forming small ponds was associated with disease in monthly rainfall were obtained from the meteorological animals in the Cerrado region. Equids usually remain for station Mestre Padre Bombled and the experimental farm long periods inside flooded areas or ponds, where they eat at the Federal University of Mato Grosso. the grass on the surface, or simply as a behavioral characteristic [4,17], increasing the chance of contact with the 2.2. Selection of the Study Population pathogen. The spatial distribution showed a predominance of properties located in the Pantanal (70.6%), followed by The descriptive observational method was used for the the Cerrado (20.6%) and ecotones (8.8%). A highly variable study of the equine population at risk between the years disease prevalence was also observed between the biomes. 2007 and 2010. Cross-sectional analyses were used to The prevalence in the Pantanal region ranged from 0.9% to evaluate the prevalence risk ratio in the herd. Analyses of 66.7%, whereas in the Cerrado, it ranged from 2.2% to 33.3%. each case were conducted because of migratory charac- An irregular distribution of the cases was noted in the herds teristics of the herd. Epidemiological data regarding the in consecutive years. This was likely to be a consequence of sex, age, species (horses or hybrids, which were composed animal migration to different areas and/or rainfall varia- mainly of mules), breed, coat color (bays, buckskins, tion. Generally, considering the total of animals exposed, chestnuts, grays or tobianos), presence of underlying the disease prevalence was approximately 5% in both diseases, vaccination, and previous treatment approaches biomes (Table 1). were assessed for each animal enrolled. Clinical data for the Infection by P. insidiosum was more prevalent in adult site (axial or appendicular) and presentation of the lesions animals (>1 year old). The relative risk of adult animals (unifocal or multifocal), and management and outcome of versus that of animals younger than 1 year old acquiring infection were also collected. The temporal distribution of the disease was 8.07 (P < .05; 95% confidence interval [CI], cases was based on the size and aspect of the horse’s lesion 1.13-57.8, using the chi-square test; Table 2). This finding is and body condition and information from the breeders. in agreement with previous studies which indicate that equine pythiosis is more frequent in adult animals [6,18- 2.3. Diagnosis 21]. Interestingly, equine sarcoid has also been reported in adult animals between 1 and 7 years of age [22,23]. The presumptive diagnosis was based on the observation Indeed, the association between pythiosis and sarcoid has of ulcerated, granulomatous skin lesions with abundant been previously reported, showing that ulcerated lesions 272 C.E.P. dos Santos et al. / Journal of Equine Veterinary Science 34 (2014) 270–274

Table 1 Epidemiological proﬁle of equine pythiosis in the Brazilian Pantanal and Cerrado biomes between 2007 and 2010

Region No. of Properties No. of Animals Exposed Cases Deaths Prevalencea (%) Mortalityb (%) Lethalityb (%) Pantanal 24 1,201 65 15 5.41A 1.25 23.1 Cerrado 10 216 11 5 5.09A 2.31 45.5 Total 34 1,417 76 20 5.36 1.41 26.3

a Values followed by the same superscript capital letter within a column are not significantly different (P > .05; Fisher exact test). b Mortality and lethality rates were not included in the statistical analysis because of the potential bias caused by the use of immunotherapy and/or surgery. present in sarcoid tumors could favor penetration of the it was not possible to determine the results. The lethality zoospores into the host [6]. rate in nontreated animals or those empirically treated Fast-growing lesions were characteristically ulcerated, with wound debridement and cauterization by incandes- associated with the formation of extensive granular tissue cent iron reached 73.3%. Significant differences in mortality and sinuses containing kunkers, and were linked to chronic and lethality were observed among treated and untreated debilitating disease. Unifocal presentation was observed animals (P < .05), indicating that the use of immunother- in 79% of the cases (60 of 76). Multifocal lesions were apeutic products can prevent economic losses. Conversely, observed in 21% (16 of 76) of the animals and were no statistical differences in mortality and lethality were distributed concomitantly among pelvic and thoracic limbs, observed between animals treated with immunotherapy chest and limbs, and the abdomen and chest. Appendicular and those treated with the combination of immunotherapy lesions were found in 76.3% (58 of 76) of the animals, fol- and surgery. The disease was quiescent in a few cases, in lowed by axial lesions (22.4%) involving mainly the trunk agreement with previous reports [6]. Notably, the chronic (chest and abdominal regions), and one single case of evolution of pythiosis is likely to contribute to the life cycle a lesion in the cephalic region. Such a lesion distribution is of the pathogen during the drought period, once P. insidi- not surprising considering the contact of lower parts of the osum asexual reproduction occurs in aquatic environments body with the water, but once again, distribution followed [7]. Therefore, the role of equids in the maintenance of the the same pattern for sarcoid lesions, which is more often oomycete life cycle seems to be essentially important, found, in decreasing order, in the limbs, trunk, and cephalic mainly in the Pantanal area, where the extensive drought regions [22]. We highlight here the importance of the period associated with low temperatures imposes a harsh differential diagnoses between equine pythiosis and condition on the survival of the pathogen in the environ- sarcoid because of the similarities in aspect and localization ment. Once kunkers are expelled, they produce sporangia of the lesions and age of occurrence in the animals. Both in wet environments [7], but they also are able to produce ELISA and PCR assays were positive for 100% of the animals, zoospores after plant colonization [24]. showing granulomatous and eosinophilic tissue infiltrates The temporal distribution showed an increase in the associated with the presence of broad and sparsely septate number of cases from mid spring onward, reaching peaks in hyphae. Isolation of the pathogen was sometimes impaired January and March, months with the highest rainfall by the distance and time before culturing. indices in the region. The number of cases declined Mortality and lethality rates were 1.3% and 23.1%, abruptly in the drought period (April-September; Fig. 1). respectively, in the Pantanal and 2.3% and 45.5%, respec- The average maximum and minimum temperatures were tively, in the Cerrado. Importantly, these rates are likely to 33.1C and 20.5C, respectively, during the studied period. be underestimated because of the treatment with immu- According to Leal et al [6], the peak flood period between notherapy, mainly among the cases observed in the Pan- February and May is related to the highest number of cases tanal biome [3]. In most cases, the death of the animals in the southern Pantanal region. The floods in that region occurred between 3 and 7 months after the lesion occur after those in the northern region, as a result of the appeared, in agreement with previous reports [19-21]. water flowing from the north to the south. Therefore, the Shorter clinical courses were observed in animals coin- greatest occurrence of cases occurring during the rainy fected with equine infectious anemia virus, Trypanosoma period gives the popular name to the disease, “ferida da spp, or Conidiobolus spp or with chronic lesions that had moda,” which is Portuguese for “trend wound.” In India, the secondary bacterial infection. Data from the 62 cases observed in our study are presented in Table 3. In 13 cases, Table 3 Efficacy of different strategies used to treat equine pythiosis in the Bra- Table 2 zilian Pantanal and Cerrado biomes between 2007 and 2010 Age-related risk of equine pythiosis observed in the Brazilian Pantanal and Cerrado biomes between 2007 and 2010 Treatment Cure Death Total Efficacya A Age Pythiosis Total Immunotherapy 27 7 34 79.4 Immunotherapy and surgery 11 2 13 84.6A Yes No Empiricalb 4 11 15 26.7B >1 year 75 1,204 1,279 Total 42 20 62 <1 year 1 137 138 a Values followed by different superscript capital letters within Total 76 1,341 1,417 a column are statistically different (P < .001; Fisher exact test). The relative risk of equine pythiosis among adult animals was 8.09 (95% b Procedures adopted by nonveterinarians, such as the use of incan- CI, 1.13-57.8) compared to <1-year-old animals. descent iron, acids, and different local and systemic drugs. C.E.P. dos Santos et al. / Journal of Equine Veterinary Science 34 (2014) 270–274 273

No signiﬁcant differences were observed between sexes. Thirty males (39.5%) and 46 females (60.5%) were affected, differing from previous studies in the Brazilian Pantanal and Egypt, where 81% and 87.8% of the cases were diagnosed in females, respectively [6,18]. Horses were more affected (97.4%) than domestic equine hybrids (2.6%). Such differences are likely to be related to the great predominance of horses (96.2%) compared to hybrids (3.8%) in the region [25]. Therefore, both species seem to be equally susceptible to the pathogen. The same pattern was found in relation to the breed of the animals. The disease affected 54% of nonbreed horses, 44.7% of horses of the Pantaneira breed, and 1.3% of the Paint Horse breed. The Pantaneira Fig. 1. Correlation between temporal distribution of equine pythiosis cases breed accounts for most of the animals in the area. and the monthly average rainfall in the Pantanal and nearby areas from Gray-colored horses are predominant in the Pantanal 2007-2010 (R2 ¼ 0.775; P < .001). region. However, bay-colored animals were more frequently infected by P. insidiosum (34.2%), followed by disease is called “bursattee,” a term derived from “burus,” buckskins (26.3%), grays (19.7%), chestnuts (14.5%), and a word signifying rain [7]. tobianos (5.3%). To our knowledge, this is the ﬁrst report of The rise of cases in the northern region immediately the importance of equine coat color in equine pythiosis. We after the beginning of the rainy period, with peaks observed an increased frequency of lesions in dark- following the same temporal tendency, suggests a short pigmented horses (73.7%) in comparison to lightly pig- incubation period, between 15 and 30 days, as estimated mented horses (15.8%). This was strikingly apparent in previously [17]. Temperatures above 30C are also favorable tobiano horses, in which the lesions were present in pig- for the development of zoospores, and, although the cases mented areas or in the transition between white and occur during the whole year, outbreaks are often reported colored coat areas (Fig. 2). Hematophagous insects prefer in warm, rainy summers. dark-pigmented areas for blood feeding and are

Fig. 2. Anatomical distribution of the lesions in horses with pythiosis. Note the occurrence of lesions in dark pigmented areas or in light-to-dark skin color transition. 274 C.E.P. dos Santos et al. / Journal of Equine Veterinary Science 34 (2014) 270–274 hypothesized to be important from an epidemiological [6] Leal ABM, Leal AT, Santurio JM, Kommers GD, Catto JB. Equine point of view. First, insect bites could favor the penetration pythiosis in the Brazilian Pantanal region: clinical and pathological findings of typical and atypical cases. Pesqui Vet Bras of zoospores into the injured skin, and, second, infected 2001;21:151–6. mosquitoes could transmit the disease directly, as P. insid- [7] Mendoza L, Hernandez F, Ajello L. Life cycle of the human and iosum has been identified in tropical mosquito larvae [1,26]. animal oomycete pathogen Pythium insidiosum. J Clin Microbiol 1993;31:2967–73. No predisposing factors in animals have been reported so far. [8] Mendoza L, Ajello L, Mcginnis MR. Infections caused by the However, reinfection is commonly observed in successfully oomycetous pathogen Pythium insidiosum. J Mycol Med 1996;6: treated horses [3]. Insect-transmitted diseases such as equine 151–64. [9] Loreto ES, Alves SH, Santurio JM, Nogueira CW, Zeni G. Diphenyl infectious anemia and trypanosomiasis are endemic in the diselenide in vitro and in vivo activity against the oomycete Pythium Brazilian Pantanal, but their correlation to pythiosis remains insidiosum. Vet Microbiol 2011;156:222–6. uncertain and deserves further investigation. In humans, the [10] Dória RG, Freitas SH, Linardi RL, Mendonça FdeS, Arruda LP, Boabaid FM, et al. Treatment of pythiosis in equine limbs using vascular and disseminated forms of pythiosis are related to iron- intravenous regional perfusion of amphotericin B. Vet Surg 2012; overloaded, thalassemic patients [5].Irondeficiency is 41:759–65. uncommon in horses, with the exception of foals, because of the [11] Pires L, Bosco SMG, Silva NF, Kurachi C. Photodynamic therapy for low iron levels in milk. Indeed, ferritin levels are usually elevated pythiosis. Vet Dermatol 2013;24:e130. [12] Zanette RA, Alves SH, Pilotto MB, Weiblen C, Fighera RA, Wolkmer P, in adult horses [27], and the iron content present in the soil and et al. Iron chelation therapy as a treatment for Pythium insidiosum in forage in the Pantanal region is high [28]. This is in agreement an animal model. J Antimicrob Chemother 2013;68:1144–7. with the higher prevalence of the disease in adult horses in [13] Pereira DIB, Santurio JM, Alves SH, Argenta JS, Cavalheiro AS, Ferreiro L. In vitro zoosporogenesis among oomycetes Pythium comparison to that in animals <1yearold,observedinour insidiosum isolates. Cienc Rural 2008;38:143–7. study. In rabbits, the disease causes microcytic hypochromic [14] Botton SA, Pereira DIB, Costa MM, Azevedo MI, Argenta JS, Jesus FPK, fi anemia and the depletion of bodily iron stores [29]. Therefore, et al. Identi cation of Pythium insidiosum by nested PCR in cutaneous lesions of Brazilian horses and rabbits. Curr Microbiol 2011; theroleofironintheinfectioninhorsescannotberuledout. 62:1225–9. [15] Dean AG, Dean JA, Coulombier D, Brendel KA, Smith DC, Burton AH, 4. Conclusions et al. Epi Info version 6: a word processing, database, and statistics program for epidemiology on microcomputers. Atlanta, GA: Centers for Disease Control and Prevention; 1994. The epidemiological survey of equine pythiosis in the [16] Miller RI, Campbell RSF. Clinical observations on equine phycomy- Brazilian Pantanal and nearby areas of Cerrado indicated cosis. Aust Vet J 1982;58:221–6. a prevalence of approximately 5%. Cases were intrinsically [17] Marcolongo-Pereira C, Sallis ESV, Raffi MB, Pereira DIB, Hinnah FL, Coelho ACB, et al. Epidemiology of equine pythiosis in correlated to the season of the year and rainfall indices. southern of Rio Grande do Sul State, Brazil. Pesqui Vet Bras 2012; Unifocal lesions in the limbs were predominant and were 32:865–8. similar to sarcoid tumors in aspect and anatomical locali- [18] Mosbah E, Karrouf GIA, Younis EA, Saad HS, Ahdy A, Zaghloul AE. zation and the animal’s age at which they occurred. Diagnosis and surgical management of pythiosis in draft horses: report of 33 cases in Egypt. J Equine Vet Sci 2012;32:164–9. Moreover, the animals that had a higher chance of [19] Alfaro AA, Mendoza L. Four cases of equine bone lesions caused by acquiring the disease were those older than 1 year and Pythium insidiosum. Equine Vet J 1990;22:295–7. [20] Headley SA, Arruda HN. Equine cutaneous pythiosis: a report of four mainly those with dark coat color or with dark-pigmented – fi cases. Cienc Rural 2004;34:289 92. areas. This nding is interesting from an epidemiological [21] Sallis ESV, Pereira DJB, Raffi MB. Cutaneous pythiosis in horses: 14 point of view especially because these anatomical areas are cases. Cienc Rural 2003;33:899–903. also the preferred feeding sites of hematophagous insects. [22] Brum JS, Souza TM, Barros CSL. Epidemiological aspects and anatomic distribution of equine sarcoid in the state of Rio Grande do Sul, Brazil: 40 cases. Pesqui Vet Bras 2010;30:839–43. Acknowledgments [23] Scott DW, Miller WH. Neoplastic and non-neoplastic tumors. In: Scott DW, Miller WH, editors. Equine dermatology. St. Louis, MO: WB Saunders; 2003. p. 719–31. The authors thank CAPES and FAPERGS for granting [24] Corrêa BF, Pizoni C, Mendes JF, Nonnemacher DVF, De Oliveira MP, predoctoral scholarships to C. E. P. S. and R. A. Z., respectively, Pereira DIB. Importância dos kunkers na manutenção do ciclo biológico – and FAPEMAT for project grant 002.282/2007. do oomiceto Pythium insidiosum resultados preliminares [Importance of kunkers on the maintenance of Pythium insidiosum life cycle– preliminary data]. In: Proceedings of the Congresso de Iniciação References Científica. Universidade Federal de Pelotas, Pelotas, Brazil; 2009 [in Portuguese]. http://www.ufpel.tche.br/cic/2009/cd/pdf/CB/CB_00303. [1] Gaastra W, Lipman LJ, De Cock AW, Exel TK, Pegge RB, pdf [accessed 02/09/2012]. Scheurwater J, et al. Pythium insidiosum: an overview. Vet Micro- [25] Brazilian Institute of Geography and Statistics. 2006 Agricultural biol 2010;146:1–16. Census. Rio de Janeiro: IBGE; 2007 [in Portuguese]. [2] Pesavento PA, Barr B, Riggs SM, Eigenheer AL, Pamma R, Walker RL. [26] Schurko AM, Mendoza L, De Cock AWAM, Klassen GR. Evidence for Cutaneous pythiosis in a nestling white-faced ibis. Vet Pathol 2008; geographic clusters: molecular genetic differences among strains of 45:538–41. Pythium insidiosum from Asia, Australia and the Americas are [3] Santos CEP, Marques LC, Zanette RA, Jesus FPK, Santurio JM. Does explored. Mycologia 2003;95:200–8. immunotherapy protect equines from reinfection by the oomycete [27] Harvey JW, Asquith RL, Sussman WA, Kivipelto J. Serum ferritin, Pythium insidiosum? Clin Vaccine Immunol 2011;18:1397–9. serum iron, and erythrocyte values in foals. Am J Vet Res 1987;48: [4] Santurio JM, Alves SH, Pereira DB, Argenta JS. Pythiosis: an emergent 1348–52. mycosis. Acta Sci Vet 2006;34:1–14. [28] Machado LP, Kohayagawa A, Yonezawa LA, Silveira VF, Saito ME. [5] Krajaejun T, Sathapatayavongs B, Pracharktam R, Nitiyanant P, Iron metabolism in athletics horses. Cienc Rural 2010;40:703–11. Leelachaikul P, Wanachiwanawin W, et al. Clinical and epidemio- [29] Zanette RA, Bitencourt PER, Alves SH, Fighera RA, Flores MM, logical analysis of human pythiosis in Thailand. Clin Infect Dis 2006; Wolkmer P, et al. Insights into the pathophysiology of iron metabolism 43:569–76. in Pythium insidiosum infections. Vet Microbiol 2013;162:826–30.