J. Comp. Path. 2012, Vol. 146, 122e131 Available online at www.sciencedirect.com

www.elsevier.com/locate/jcpa

INFECTIOUS DISEASE

A Comparative Study of the Histopathology andImmunohistochemistry of Pythiosis in Horses,

Dogs and Cattle

Cor

002

doi

T. B. Martins*, G. D. Kommers*, M. E. Trost*, M. A. Inkelmann*,R. A. Fighera* and A. L. Schild†

*Departamento de Patologia, Universidade Federal de Santa Maria, Av. Roraima 1000, Camobi 97105-900, Santa Maria-

RS, and †Laborat�orio Regional de Diagn�ostico, Universidade Federal de Pelotas, Campus Universit�ario s/n, 96010-900,Pelotas-RS, Brazil

resp

1-99

:10.1

Summary

Twenty-one cases of pythiosis in horses (n¼ 10), dogs (n¼ 9) and cattle (n¼ 2) were investigated. The aetiologyin all cases was confirmed by immunohistochemistry. Data related to the clinical course and outcome andlocalization of the lesions were obtained from pathology reports. The equine lesions consisted of fibrotic tissuewith multiple, often coalescing, areas of immature granulation tissue encircling eosinophilic cores. Affecteddogs had gastrointestinal and/or cutaneous lesions with either or both of a granulomatous/pyogranulomatousor necrotizing eosinophilic inflammatory reaction. In cattle, cutaneous lesions were characterized bymultifocalto coalescing granulomas with surrounding fibrosis. The number of intralesional hyphae, the distribution ofhyphae, the presence of angioinvasion and the nature of the local inflammatory reactions were associatedwith the different types of lesions observed.

� 2011 Elsevier Ltd. All rights reserved.

Keywords: cattle; dog; horse; Pythium insidiosum

Introduction

Pythiosis is caused by the oomycete Pythium insidiosum.The disease affects man (Wanachiwanawin et al.,2004) and horses (Mendoza and Newton, 2005), live-stock (Miller et al., 1985; Santurio et al., 1998; Tabosaet al., 2004), dogs (Miller, 1985) and cats (Rakich et al.,2005) among other domestic and wild animal species.

The clinical presentation of pythiosis varies widelybetween species (Santurio and Ferreiro, 2008).Although the disease is potentially fatal in all species(Mendoza et al., 2003), spontaneous healing isreported in cattle (Santurio et al., 1998; Gabriel et al.,2008; Grecco et al., 2009). It has been suggested thatthe nature of the immune response to the organismmay determine whether the lesions resolve or persist(Mendoza et al., 2003; Mendoza and Newton, 2005).

ondence to: G. D. Kommers (e-mail: glaukommers@yahoo.com).

75/$ - see front matter

016/j.jcpa.2011.06.006

The aim of the present study was to evaluate com-paratively the histopathological and immunohisto-chemical aspects of pythiosis in horses, dogs andcattle.

Materials and Methods

Twenty-one cases of pythiosis in horses (n¼ 10), dogs(n ¼ 9) and cattle (n ¼ 2) were investigated.Formalin-fixed, paraffin wax-embedded sampleswere selected from the archives of the Laborat�oriode Patologia Veterin�aria, Universidade Federal deSanta Maria, Brazil, and from the Laborat�orioRegional de Diagn�ostico, Universidade Federal dePelotas, Brazil. Information related to the clinicalpresentation and outcome and the location of lesionswere obtained from the pathology reports.

Serial sections (3 mm) were cut from the tissueblocks for histochemical staining and immuno-histochemistry (IHC). The aetiology was confirmed

� 2011 Elsevier Ltd. All rights reserved.

Pythiosis in Horses, Dogs and Cattle 123

in all cases by IHC using a rabbit polyclonalanti-P. insidiosum antibody as described by Gabrielet al. (2008). Briefly, sections were dewaxed and re-hydrated and endogenous peroxidase activity wasblocked with H2O2 3% in distilled water. Antigenretrieval was by microwaving (10 min at fullpower) in TRISeEDTA (pH 9.0). Sections wereincubated at 37�C for 60 min with the primary an-tibody diluted at 1 in 1,000. The secondary reagentwas biotinylated anti-rabbit antibody (LSAB+Sys-tem-AP or -HRP; Dako Corp., Carpinteria, Cali-fornia, USA) followed by streptavidinealkalinephosphatase (LSAB+System-AP, Dako) or strepta-vidineperoxidase (LSAB+System-HRP, Dako).Substrate development was with Liquid PermanentRed (Dako) or 3, 30diaminobenzidine (DAB; Sig-maeAldrich, Saint Louis, Missouri, USA), respec-tively. Sections were counterstained lightly withhaematoxylin and coverslipped.

The specificity of the primary antibody was testedin a case of canine gastrointestinal pythiosis (dog 3,

Table

Animal data, clinical forms of disease, location

Species Animal Breed Sex Age (years) Clinical presentatio

Equine 1 NR NR NR Cutaneous/SC

2 Mixed F 7 Cutaneous/SC

3 Thoroughbred F 12 Cutaneous/SC4* Mixed M 2 Cutaneous/SC

5 Mixed F 9 Cutaneous/SC6 NR M Adult Cutaneous/SC

7* NR NR NR Cutaneous/SC

8 Crioula F 15 Cutaneous/SC9 Crioula F 17 Cutaneous/SC

10 Crioula F 7 Cutaneous/SC

Canine 1 Labrador M 3 Gastrointestina2 Mixed M 1.5 Gastrointestina

3* Mixed F 3 Gastrointestinal

Cutaneous

4 Mixed M 1 Gastrointestina

5 Shar-pei F 2 Gastrointestina

6 NR M 1.5 Gastrointestina7 Labrador F 1.5 Gastrointestinal

Cutaneous

8 German

shepherd dog

M 5 Cutaneous

9 Boxer M 2 Cutaneous

Bovine 1 Mixed NR Up to 3 Cutaneous

2 Mixed M 2 Cutaneous

NR, not recorded; F, female; M, male; SLN, submandibular lymph node*P. insidiosum was isolated in microbiological culture.†Tissue analysed in this study.

Table 1) that was confirmed by microbiologicalculture and zoosporogenesis (Rech et al., 2004). Theanti-P. insidiosum antibody was also tested by IHCwith samples of tissue from known cases of zygomycosis(bovineandavian), aspergillosis (avian), candidiasis (fe-line) and cryptococcosis (equine). All of these reactionswere negative, supporting the specificity of the reagent.

Serial sections were stained with haematoxylinand eosin (HE), Grocott’s methenamine silver stain(GMS), periodic acideSchiff (PAS), sirius red (SR)and toluidine blue (TB). The SR technique, usedto demonstrate eosinophil granules, was slightlymodified from Wehrend et al. (2004). A combina-tion of GMS and SR (GMSeSR) was used inselected cases to demonstrate intralesional hyphaeand eosinophils.

Each technique was employed to evaluate a distinctmorphological aspect and a comparative analysis ofserial fields was made. HE was used to evaluate themorphology, localization and extent of the lesionsand the type of inflammatory response. The intensity

1

of the lesions and clinical outcome of cases

n Location Outcome Previous report

NR NR

Abdomen NR

Abdomen NRLip Humane

destruction

Jaw NRLip† and SLN Humane

destruction

NR NR

Abdomen NRLeft hindlimb Humane

destruction

Lip NR

l MLN Death Trost et al., 2009l Stomach† and MLN NR Trost et al., 2009

/ Colon†/lip Humane

destruction

Rech et al., 2004;

Trost et al., 2009

l Stomach and smallintestine†

Humanedestruction

Trost et al., 2009

l Mesentery† and MLN Death Trost et al., 2009

l Large intestine Death/ Large intestine/thorax Death Pereira et al., 2010

Foreskin†, penis and

scrotum

NR

Multifocal Death

Multifocal Spontaneous

healing

Gabriel et al., 2008

Multifocal Spontaneoushealing

Greco et al., 2009

; LHL, left hindlimb; MLN, mesenteric lymph node.

124 T.B. Martins et al.

of the inflammatory response was graded as absent,mild (sparse, well-dispersed cells), moderate (scat-tered, but limited aggregates of inflammatory cells)or severe (diffuse sheets of inflammatory cells). GMSwas used to analyse the quantity, distribution andmorphological features of hyphae (through whichhyphal viability was inferred) and angioinvasion bythe agent. Eosinophils and patterns of theSplendore-Hoeppli reaction were evaluated throughSR staining. PAS and TB were used to evaluatehyphae and mast cells, respectively.

Results

Clinical data are summarized in Table 1. With theexception of the cattle, the cases were isolated incidentsof pythiosis.The cattlewere part of twooutbreaks of cu-taneous pythiosis. In all cases, fungal hyphae werestrongly labelled by the P. insidiosum-specific antibody(Figs. 1aec).

Fig. 1. (a) Equine cutaneous pythiosis. Hyphae of P. insidiosum atthe periphery of a kunker. IHC. Bar, 20 mm. (b) Caninegastrointestinal pythiosis. Hyphae of P. insidiosum in thecentre of a pyogranuloma. IHC. Bar, 30 mm. (c) Bovine cu-taneous pythiosis. Hyphae of P. insidiosum in the centre ofa pyogranuloma. IHC. Bar, 30 mm.

Equine Pythiosis

Equine skin sections were extensively ulcerated withsurface fibrin, neutrophils and bacterial colonies.The majority of the samples comprised of fibrous con-nective tissue extending from the dermis to the subcu-tis. There were numerous, often coalescing, foci ofimmature granulation tissue surrounding eosinophilicmaterial (Fig. 2a). Grossly, sinus tracts filled bydetachable tubular structures were described. Theselatter were yellow, firm and had an irregular surface,resembling coral. These structures are classicallytermed ‘kunkers’ (Miller and Campbell, 1984; Lealet al., 2001) and this term will be used herein todescribe these structures microscopically.

Microscopically, the kunkers consisted of dense col-lections of eosinophils with numerous and poorlydefined cell nuclei and sparse hyaline trabeculae ofcollagen (collagenolysis) (Fig. 2b). The kunkerswere often surrounded by neutrophils and sat withina tract in the tissue, which separated them from adja-cent granulation tissue. Negatively-stained hyphalprofiles were present at the periphery of the kunkers.Rarely, similar smaller foci, consistent with an earlystage of kunker formation, were noted (Fig. 2c). Adegenerate artery (small or medium sized) was almostalways observed centrally within the kunker, butthere was no evidence of angioinvasion.

Rare aggregates of macrophages were presentwithin the granulation tissue. A marked infiltrationof eosinophils was present around the kunkers and oc-casional lymphocytes and plasma cells were present inthese areas. Less frequent findings were small arterialthrombi and focal areas of dermal oedema.

With GMS staining, hyphae were seen to belocated predominantly at the outer edge of thekunkers and fewer hyphal elements were scatteredthroughout the kunkers. The hyphae were broad

Fig. 2. Equine cutaneous pythiosis. (a) Dermis thickened by fibrous connective tissue encircling areas of granulation tissue with eosino-philic cores (grossly corresponding to kunkers). HE. Bar, 25 mm. (b) Kunker composed of a dense collection of eosinophils withpoorly defined cell nuclei and sparse hyaline collagen trabeculae. HE. Bar, 120 mm. (c) Early stage of kunker formation, withnumerous eosinophils. SR. Bar, 50 mm. (d) Hyphae confined to the periphery of a kunker. The hyphae are broad and sparsely sep-tate with smooth and almost parallel walls, with inconspicuous dilations. GMS. Bar, 15 mm. (e) Close relationship between hyphae(dark brown) and eosinophils (red) at the outer edge of a kunker. Eosinophils converge from peripheral tissues to degranulate overthe hyphae.GMSeSR.Bar, 20mm. (f) Bright red granular halo aroundhyphae (SH-like reaction).TheSH-likematerial consists ofmultiple SR-positive granules aggregated over the surface of the hyphae. SR. Bar, 10 mm.

Pythiosis in Horses, Dogs and Cattle 125

and sparsely septate and had smooth, almost parallelwalls with inconspicuous dilations (Fig. 2d). Horseshad a moderate (eight of 10 cases) or large (two of10 cases) number of hyphae.

The kunkers were deeply stained by SR. The mostintense staining was of clusters of intact or degranu-lated eosinophils surrounding hyphae at the peripheryof the kunkers. This relationship was clear when theGMS and SR fields were analysed comparativelyand particularly with the GMSeSR stain (Fig. 2e).

Tissue infiltration by eosinophils was also moreevident following SR staining. A deeply eosinophilic,granular halo was seen around many hyphae. Thisfinding was considered to be a reaction similar to theSplendore-Hoeppli (SH-like) reaction. Under oil im-mersion (�100 objective), the SH-like material con-sisted of multiple SR-positive granules aggregatedover the surface of the hyphae (Fig. 2f).

There were few mast cells associated with thekunkers (0e8 per high power field; HPF; �40

Fig. 3. Canine pythiosis. (a) Skin. Necro-eosinophilic pattern. Focal area of necrosis with cell debris, collagenolysis and variable numbersof eosinophils (similar to the initial stages of kunkers in horses)with intralesional negatively-stainedhyphal profiles.HE.Bar, 30mm.(b) Skin. Granulomatous pattern. Discrete granuloma composed of epithelioid macrophages and Langhans giant cells surroundedby a thin capsule of connective tissue. HE. Bar, 30 mm. (c) Large intestine. Necro-eosinophilic pattern. Hyphae are numerous andstrongly stained. They are morphologically normal, sometimes long, with smooth and almost parallel walls and regular diameters.GMS. Bar, 20 mm. (d) Mesentery. Granulomatous pattern. Hyphae are confined to granulomas and are morphologically disinte-grated (small, irregular and tortuous fragments) and, at times, less argyrophilic. GMS. Bar, 20 mm. (e) Large intestine. Necro-eosinophilic pattern (left) adjacent to granulomatous pattern (right). A comparison between the number of eosinophils and hyphalmorphology (integrity) is readily made: hyphae are normal amongst eosinophils (necro-eosinophilic pattern) and disintegratedwhen associated with giant cells (granulomatous pattern). GMSeSR. Bar, 30 mm. (f) Large intestine. Necro-eosinophilic pattern.Some hyphae are surrounded by strongly sirius red-stained granules, frequently forming a red ‘sleeve’ characteristic of the SH-likereaction. SR. Bar, 10 mm.

126 T.B. Martins et al.

objective) and a variable number within the sur-rounding granulation tissue (mean 20 per HPF;range 0e50 per HPF). There was a moderatequantity of intracytoplasmic granules in thesemast cells.

Canine Pythiosis

Two main inflammatory patterns were seen in thecanine lesions, either as sole patterns or in combina-tion. The first pattern (necro-eosinophilic) was char-acterized by broad zones of eosinophilic necrosis, cell

Pythiosis in Horses, Dogs and Cattle 127

debris, collagenolysis and variable numbers of eosino-phils (Fig. 3a). Some areas of necrosis were similar tothe early form of kunkers described in horses. The sec-ond pattern (granulomatous) consisted of epithelioidmacrophages and Langhans giant cells (GCs) mixedin different proportions. These generally formed dis-crete foci, which, in some cases, were surrounded bythin capsules of connective tissue (Fig. 3b). Rarely,granulomas with necrotic cores and surrounding lym-phocytes and plasma cells were present. Negatively-stained hyphal profiles were seen within the

Fig. 4. Bovine cutaneous pythiosis. (a) Discrete granuloma composed oa delicate connective tissue capsule.HE. Bar, 30mm. (b)Discretmorphonuclear leucocytes. HE. Bar, 50 mm. (c) Classical SH retours, encircling hyphae. Note the close relationship between thsilver-stained hyphae are confined to the centre of a granulomared-stained eosinophils inside coalescing pyogranulomas. FewSH-like reaction surrounding a hyphum. There are degranula10 mm.

necro-eosinophilic lesions and some hyphae wereweakly basophilic. Sporadically, a mild multifocalgranulomatous response with vacuolated macro-phages and few GCs was seen adjacent tonecro-eosinophilic areas. Combined foci of both re-sponses were considered pyogranulomatous in nature.

In the gastrointestinal tract, lesions affected one toall layers, mainly the submucosa andmuscularis prop-ria. In the cutaneous form, lesions extended from theepidermis, which was usually ulcerated, to the hypo-dermis. In four of nine cases, therewas a predominance

f epithelioidmacrophages andLanghans giant cells surrounded bye pyogranulomawith amoderate number of centrally located poly-action consisting of lightly eosinophilic material with irregular con-e hyphum and the Langhans giant cell. HE. Bar, 20 mm. (d) Sparseand are largely disintegrated. GMS. Bar, 50 mm. (e) Strongly siriusneutrophils are present among the eosinophils. SR. Bar, 50 mm. (f)ted eosinophils immediately around the SH-like reaction. SR. Bar,

128 T.B. Martins et al.

of the granulomatous pattern over the necro-eosinophilic pattern (two gastrointestinal, one cutane-ous and one affecting lymph nodes). Three dogs hada predominant necro-eosinophilic pattern (twogastrointestinal and one cutaneous). Dog 7 had apredominantly granulomatous response in the gastro-intestinal tract and a predominantly necro-eosinophilic pattern in the skin. In dog 5, only thegranulomatous pattern was seen, and this was re-stricted to the gastrointestinal tract. Less frequent find-ings included fibrinoid necrosis of arterial walls(especially in the lymph node of dog 1) and small arte-rial thrombi.

In dogs, the hyphal morphology following GMSstaining varied according to the type of local inflam-matory response. In the necro-eosinophilic areas,hyphae were numerous, strongly GMS positive andmorphologically normal (long, with smooth andalmost parallel walls and regular diameters)(Fig. 3c). However, hyphae confined to granulomasor inside GCs were less numerous, disintegrated(small, irregular and tortuous fragments) and, attimes, less argyrophilic (Fig. 3d). Hyphae were sparsein areas where only the granulomatous pattern wasobserved (e.g. dog 5). Mild angioinvasion was notedrarely.

Amoderate number of eosinophils were evident fol-lowing SR staining and these were primarily locatedin the necro-eosinophilic areas. In areas of granulo-matous inflammation, the eosinophils were randomlyplaced and intact. The comparison between eosino-phils and hyphal morphology was readily establishedusing the GMSeSR stain (Fig. 3e). The number ofmast cells varied in both types of lesion and these cellswere generally absent or sparse (1e5 per HPF) insideand around the lesions. Similar to horses, some hy-phae were surrounded by strongly SR-positive gran-ules that often formed the ‘red sleeve’ characteristicof an SH-like reaction (Fig. 3f).

Bovine Pythiosis

The lesions in both animals (each from a differentherd) were indistinguishable. The main feature wasthe presence of multifocal to coalescing discretegranulomas surrounded by dermal collagen. Twovariants of granulomas were noted. The first consistedof a centre of epithelioid macrophages and/or Lan-ghans GCs surrounded by a thin zone of vacuolatedmacrophages and a delicate connective tissue ‘cap-sule’ as an outer layer (Fig. 4a). The second variantwas similar, but in addition there was a mixture of eo-sinophils and neutrophils within the centre of thelesions (pyogranulomas) (Fig. 4b). Within both typesof granulomas, negatively-stained hyphal profileswere seen.

Of the few hyphae in the centres of the pyogranulo-mas, some were surrounded by the SH-like reaction,which formed a thick-fringed halo. The classical SHpresentation, consisting of lightly eosinophilic andirregular contours, was rarely seen, but alwaysencircled hyphae inside granulomas. Interestingly,the SH-like reaction was surrounded by eosinophils,whereas the classical SH reaction was closely associ-ated (through phagocytosis) with macrophages andGCs (Fig. 4c).

Hyphae in cattle were confined to granulomas.They were sparse and morphologically disintegratedwhen seen byGMS staining (Fig. 4d), except for thosesurrounded by the SH or SH-like reaction. Angioin-vasion was not detected. Using the SR stain, it wasclear that the centres of the pyogranulomas were com-posed almost exclusively of eosinophils, althougha small number of neutrophils were also present(Fig. 4e). Under oil immersion, degranulated eosino-phils were noted immediately surrounding theSH-like reaction (Fig. 4f). Conversely, the SH reac-tion did not stain with SR. Similar to horses anddogs, the hyphae were consistently PAS negative incattle.

Few mast cells (2e3 per HPF) were seen and thesewere mostly around blood vessels surrounding thegranulomas. Scattered granules were present in thecytoplasm of these cells.

Discussion

Although a disparate number of cases were studiedfrom each species, which reflects the higher preva-lence of equine (frequent) over canine (occasional)and bovine (rare) pythiosis in Southern Brazil, impor-tant comparative morphological aspects were identi-fied in this study.

The lesions of equine cutaneous pythiosis are oftendescribed as areas of granulomatous inflammation(Miller and Campbell, 1984; Mendoza and Alfaro,1986; Ginn et al., 2007); however, the cellularcomponents characteristic of this type ofinflammation were not recognized in equine lesionsin this study. Therefore, the lesion was classified asan eosinophilic dermatitis and panniculitisassociated with the formation of granulation tissue.

Three similar equine diseases (i.e. pythiosis, basi-diobolomycosis and conidiobolomycosis) are some-times collectively called phycomycosis (Miller andCampbell, 1982). This could explain the general useof the term ‘granulomatous’, which better describesthe lesions caused by zygomycetes, to characterizealso the infection caused by P. insidiosum.

Mast cells were more often observed in the equinelesions. Mast cells and eosinophils participate in

Pythiosis in Horses, Dogs and Cattle 129

a self-perpetuating complex cycle (Munitz and Levi-Schaffer, 2004). The higher number of mast cellswithin the inflammatory infiltrates, and the constanteosinophil influx located around them, may havebeen the cause of formation of the kunkers in theequine lesions.

Two distinct inflammatory patterns were observedin the canine lesions: granulomatous (or sometimespyogranulomatous) and necro-eosinophilic. Similarpatterns of inflammation have been described ina case study of 60 dogs with gastrointestinal phycomy-cosis, including pythiosis (Miller, 1985). However, inmany reports of canine pythiosis, inflammation isclassified only as granulomatous and/or pyogranu-lomatous (Fischer et al., 1994; Helman and Oliver,1999; Hensel et al., 2003).

The inflammatory reactions observed in cattle weresimilar to those described in the sparse literature onbovine pythiosis. Histological presentations includepyogranulomas with a core of eosinophils and neutro-phils (Santurio et al., 1998), granulomas with inconsis-tent central caseous necrosis (Miller et al., 1985) andmultifocal areas of necrosis with eosinophils, neutro-phils, plasma cells, macrophages and GCs (P�erez et al.,2005). Hyphae have been reported inside GCs andhair follicles and distributed in necrotic areas (Milleret al., 1985; Santurio et al., 1998; P�erez et al., 2005).

In this study, only the bovine lesions had two pat-terns of the SHphenomenon; one strongly SR positiveand closely associated with the presence of eosinophilsand a second that was SR negative and closely relatedto the presence of macrophages and GCs. The SRstain was particularly useful for quantifying and local-izing the eosinophilic response in all species and it alsoshowed the participation of eosinophil granules in theprocess described as SH-like (Mendoza and Alfaro,1986).

The SH reaction is described in human conjuncti-val lesions associated with the presumed migrationof helminth larvae. Immunohistochemically, thesehuman SH reactions have two distinct patterns oflabelling: the first reveals the presence of immuno-globulins, whereas the second reveals mainly eosino-phil major basic protein (MBP) surrounding theaetiological agent. This variation is due to a numberof factors (Read et al., 2005), but differences in proteincomposition could explain the different morphologi-cal patterns of SH noted in the bovine lesions of thepresent study.

Hyphae were not stained by the PAS reaction inany of the lesions studied. The PAS stain, widely uti-lized for identification of fungi, does not usually stainthe hyphae of P. insidiosum (Grooters, 2003). Thisoomycete does not produce chitin as a constituent ofthe cell wall (Alexopoulos et al., 1996) and chitin is

the main component of fungal organisms that isstained by the PAS technique (Culling et al., 1985).

Horses had the most lesional hyphae, followed bydogs and then cattle. In horses, the hyphae were con-fined to kunkers and morphologically appeared to beviable. It is worth noting that kunkers are generallyused for culturing the agent due to the viability ofthe hyphae within them (Mendoza, 1987). The abun-dant eosinophilic response in horses, in which the dis-ease is progressive and fatal, has been consideredineffective in combating and eliminating P. insidiosum

hyphae (Mendoza et al., 2003).In dogs, the number and morphology of hyphae

varied according to the type of inflammatoryresponse. Hyphae were more abundant and morpho-logically viable in necro-eosinophilic lesions andin most cases were sparse and disrupted inside granu-lomas. Such irregular quantitative distribution asso-ciated with the type of inflammation has alreadybeen described in canine phycomycosis (Miller,1985); however, the author did not correlate the dif-ferences in hyphal morphology with the type ofinflammatory response, as described here.

In cattle, hyphae were sparse and were limited togranulomas/pyogranulomas in which they displayedevidence of disruption. The loss of hyphal integritywas often observed in dogs and cattle in associationwith a granulomatous response. It is worth notingthat a self-limiting clinical course was observed inthree out of five reports of bovine cutaneous pythiosis(Santurio et al., 1998; Gabriel et al., 2008; Grecco et al.,2009), including all the affected cattle in the twooutbreaks (n ¼ 92) of which the two animals in thepresent study are representative.

One of the most intriguing aspects of pythiosis indifferent species is the variable frequency of vascularinvasion. This feature is particularly remarkable inhuman pythiosis, which in some cases progresses to se-vere vasculitis and thrombosis and may culminate ingangrene and consequent limb amputation or radicalsurgical excision of affected areas (Wanachiwanawinet al., 2004).

Mild angioinvasionwas seen only in some of the dogsof the present study. Nevertheless, the constant pres-ence of a degenerate central artery in the equinekunkers, as previously reported (Miller andCampbell, 1984), could suggest that hyphae utilize vas-cular walls to disseminate short distances in equine cu-taneous pythiosis. In the present study, hyphae wereconfined tokunkers andwere foundprimarilyat thepe-riphery of these lesions and were not seen to spread be-tween kunkers. Invasion of the vascular wall reportedin a cat with gastrointestinal pythiosis did not resultin distant dissemination of the lesions (Rakich et al.,2005), which may justify the hypothesis given above.

130 T.B. Martins et al.

Based on the microscopical aspects of pythiosis de-scribed in the three animal species studied in thisinvestigation, the differences in clinical coursebetween the species may, at least in part, be attrib-uted to the nature of the inflammatory responsesmade to the organism. Although it has been hypoth-esized that the genetic diversity of P. insidiosum couldbe implicated in causing clinical differences amongaffected animal species, an association of specificityamong the phylogenetic groups of P. insidiosum andthe geographical regions or hosts has not been estab-lished (Chaiprasert et al, 2009).

Acknowledgments

The authors acknowledge the Conselho Nacional deDesenvolvimento Cient�ıfico e Tecnol�ogico (CNPq),Brazil, for the financial support (Grant # 478609/2010-8). G. D. Kommers has a fellowship fromCNPq (PQ-2 # 305337/2010-6) and T. B. Martinshad a scholarship from the Coordenac~ao de Aperfei-coamento de Pessoal de N�ıvel Superior (CAPES).

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½ R

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eceived, January 12th, 2011

ccepted, June 18th, 2011

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