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J. Comp. Path. 2006,Vol.134, 336^346

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Urinary Bladder Lesions inBovine Enzootic Haematuria

T. Carvalho, C. Pinto* and M. C. Peleteiro

CIISA, Faculdade deMedicinaVeterinaŁ ria, UniversidadeTeŁ cnica de Lisboa, Avenida da UniversidadeTeŁ cnica, 1300-477 Lisboa, and*Servic-o de DesenvolvimentoAgraŁ rio de Sa� oMiguel, Quinta de S Gonc-alo, 9504-541Ponta Delgada, Ac-ores, Portugal

Summary

In cattle, bracken fern chronic toxicity is characterizedby the presence of multiple tumours in thebladder (bovineenzootic haematuria). From October 1999 to March 2003, 433 urinary bladders with macroscopical lesions werecollected in the slaughterhouse of Sa� o Miguel Island (Azores, Portugal), an endemic area where Pteridium aquili-

num infestation in pastures is high. Bladder lesions were divided into three main categories (in£ammatory lesions,non-neoplastic epithelial abnormalities and tumours) and described in detail. In some cases, neoplastic growthwas con¢ned to a single site, but in most cases multiple tumours developed within the same bladder. Epithelialtumours alone were present in 51 �2% of the a¡ected bladders, mesenchymal tumours alone in 17 �4%, and bothepithelial and mesenchymal tumours in the remaining 31 �4%. The large number of tumours examined (870)revealed new categories not yet included in other veterinary classi¢cation systems, namely, inverted papilloma,papillary neoplasm of apparent low malignant potential, and haemangioendothelioma.

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Keywords: bovine enzootic haematuria; bracken fern; cattle; tumour; urinary bladder

Introduction

Naturally occurring urinary bladder neoplasmsare common in dogs, accounting for 0.5^1% of allcanine neoplasms (Maxie, 1993; Meuten, 2002;Meuten et al., 2004), and generally thought to berare in other domestic and laboratory animals(Yoshikawa et al., 1981), except in cattle. In certainparts of the world, the prevalence of bovine bladderneoplasia is extremely high, being associated withthe chronic ingestion of bracken fern (mainly Pteridiumaquilinum) (Maxie, 1993; Ozkul and Aydin, 1996;Smith, 1997; Meuten, 2002; Borzacchiello et al., 2003a;Meuten et al., 2004).Bracken fern is among the ¢ve commonest plants in

the world and is the only higher plant known to causecancer naturally in animals (Evans et al.,1972; Smith et

al., 1988; Smith, 1997). It has a considerable number oftoxic components, one of which is ptaquiloside (PT), aglycoside sesquiterpenoid capable of inducing clasto-genesis in cell cultures and also with mutagenic andcarcinogenic activity (Smith, 1997; Alonso-Amelot

front matter2006.01.001

and Avendano, 2002). PT is the main bracken carcino-gen and is carcinogenic in experimental animals (Pa-mukcu et al., 1978; Hirono et al., 1987; Shahin et al.,1998a). PT is eliminated in the urine, inducing bladdertumours in cattle, and also in the milk, with potentialrisks to human health (Evans et al., 1972; Hirono et al.,1987; Shahin et al.,1998b,1999).In cattle, bracken fern chronic toxicity causes multi-

ple tumours in the bladder wall and haemorrhagesin the bladder mucosa, giving rise to so-called bovineenzootic haematuria (BEH) (Pamukcu et al., 1976;Meuten et al., 2004). This disease, which is associatedwith haematuria, leucopenia, anaemia, and reducedhaemoglobin, has been reproduced experimentallyin bracken-fed cattle (Pamukcu et al., 1967; Prakashet al.,1996). Prakash et al. (1996) showed that, in samplesof ileum and urinary bladder, the codon 61 of h-rascarried a speci¢c mutation. DNA changes wererecently identi¢ed by Sardon et al. (2005) in bladderlesions of cattle exposed to bracken fern, H-ras expres-sion being signi¢cantly increased in chronic cystitisand tumours.

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Urinary Bladder Lesions in BEH 337

Infection by bovine papillomavirus (BPV-2) was de-monstrated by Campo et al. (1992) in bladder tumoursof cattle exposed to bracken fern. BPV-2 was isolatedfrom normal urothelium and also from naturally oc-curring and experimentally induced tumours, thevirus being capable of remaining latent in some tissues,including urinary bladder (Campo,1999). Immunosu-pression was su⁄cient to lead to premalignant lesions,but the mutagens present in bracken were responsiblefor their progression to neoplasia (Campo et al., 1992;Campo,1999; Borzacchiello et al., 2003b). Upon cancerdevelopment, BPV-2 would seemto undergo signi¢cantchanges, expressing the viral oncoprotein E5 andmod-ifying telomerase activity (Borzacchiello et al., 2003b).A similar synergism between papillomavirus andbracken fern was also postulated in bovine and humangastrointestinal tumours, this time associating BPV-4and quercetin, a mutagenic £avonoid also present inbracken fern (Campo et al.,1999).Sa� o Miguel Island, in theAzores, is an endemic area

where P. aquilinum grows in pastures. An epidemiologi-cal study between 1997 and 1999 indicated an associa-tion between BEH and the level of infestation ofpastures with P. aquilinum (Pinto et al., 2000, 2001). PTwas detected in high concentrations in P. aquilinum fromSa� o Miguel Island, where, in 1999, 21% of the dairyfarms had at least one animal with clinical signs or tu-mour lesions associated with BEH (Pinto et al., 2001).Moreover, ca 10% of the adult cows at present slaugh-tered on this island are rejected because of urinarybladder tumours (Pinto et al., 2004); due to the high in-cidence of this disease, urinary bladders are systemati-cally opened in all animals slaughtered.A newWHOclassi¢cation system for tumours of the

urinary system of domestic animals was recently pub-lished (Meuten et al., 2004). Although extensive, thisclassi¢cationwas incomplete in respect of BEH lesions.This was not surprising in view of the heterogeneityof in£ammatory lesions, epithelial abnormalities andtumours found in BEH. The aim of the present studywas therefore to perform an extensive and detailed his-tological analysis of the bladder lesions that arise inBEH.

Materials and Methods

From October 1999 to March 2003, a total of 41363Friesian cows (after calving, and42 years of age) wereslaughtered in the abattoir of Sa� oMiguel Island. Rejec-tion due to bladder lesions occurred in 5638 (13 �6%),from which 433 were selected, at random, for the pre-sent study.Their ages ranged from 2 to16 years (mean,7). Up to 90% of the animals rejected due to bladderlesions had shown haematuria at or before slaughter.

Because many bladders exhibited multiple lesions,multiple samples were collected from large and hetero-geneous tumours. In all,1337 tissue fragments were se-lected for histopathological examination. Tissues were¢xed in 10% neutral bu¡ered formalin and embeddedin para⁄n wax. Sections (3^5 mm) stained with hae-matoxylin and eosin (HE) were examined and classi-¢ed independently by two of the authors (T.C. andM.C.P.).When the diagnosis di¡ered, sections were re-examined to achieve a consensus.Histological typing of bladder tumours followed, as

far as possible,WHO established criteria for the diag-nosis of urinary bladder tumours in domestic animals(Meuten et al., 2004). However, theWHOclassi¢cationsystem for human urinary bladder tumours (Mosto¢et al.,1999; Eble et al., 2004) was used for lesions not in-cluded in the corresponding classi¢cation for domesticanimals. New categories were proposed when nomatching description was found in either of theWHOsystems.

Results

Bladder lesionswere subdivided into threemajor di¡er-ent categories, in£ammatory lesions, non-neoplasticepithelial abnormalities, and tumours.Of the 433 urinary bladders collected 373 showed

neoplasia (Fig. 1); 55 showed in£ammatory lesions ornon-neoplastic epithelial abnormalities (NNEA), orboth, NNEA being present in 26 bladders within thisgroup; and the remaining ¢ve showed no changeswhatsoever. In107 cases inwhich neoplasmswere diag-nosed, various types of NNEA were also identi¢ed inthe same organ.Bladders were considered normal when the mucosa

was lined by several (3^7) layers of normal transitional(urothelial) cells, including super¢cial, intermediateand basal cells, exhibiting characteristic polarity, withpalisading of the basal cells. Under the epithelium laythe lamina propria, displaying an interrupted layer ofsmooth muscle, consisting of the muscularis mucosae,overlying the muscularis propria (the large smoothmuscle bundles of the bladder wall).

Inflammatory Lesions

The in£ammatory lesions most frequently observedwere polypoid and follicular cystitis, both of which de-formed the bladder wall pro¢le. Polypoid cystitis wasdiagnosed when an oedematous lamina propria, oftenin£amed, gave rise to elongated mucosal folds coveredby normal urothelium (Fig. 2a). Follicular cystitis wasdiagnosed when lymphoid follicles were seen in the la-mina propria. Occasionally, reactive atypia was seen,

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* NNEA, non neoplastic epithelial abnormalities

Bladder samples

433

with neoplastic

lesions373

with no neoplastic

lesions55

with no lesions

5

neoplasmsonly 266

neoplasmsplus NNEA*

107

NNEA*(associated or not with inflammation)

26

inflammationonly 29

Fig.1. Types of histopathological lesion present in bladder samples.

Fig.2a^c. Non-neoplastic epithelial abnormalities. (a) Polypoid cystitis. (b) Cystitis cystica associatedwith cystitis glandularis (arrowhead).(c) Nephrogenic adenoma. Bovine bladder. HE. Bar,150 mm.

T. Carvalho et al.338

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Urinary Bladder Lesions in BEH 339

with variations in nuclear size and staining of transi-tional cells.

Non-neoplastic Epithelial Abnormalities

NNEA included hyperplasia, von Brunn’s nests, cystitiscystica, glandular metaplasia (cystitis glandularis andintestinal metaplasia) and nephrogenic adenoma.These lesions were often associated with neoplasia. In-creased numbers of normal transitional cell layers,either £at or undulated, were described as hyperplasia.Von Brunn nests, which were found frequently, con-sisted of compact, round aggregates of transitional cellsin the lamina propria, with or without connection tothe surface epithelium.When these aggregates showedcentral lumina, the lesionwas classi¢ed as cystitis cysti-ca (Fig. 2b). Glandular metaplasia was also frequentlyseen, characterized by mucus-containing epithelialcells of colonic type lining the surface of the bladder orforming glands in the laminapropria. Glandularmeta-plasia either resembled intestinal mucosa (‘‘intestinal

Fig.3a^c. Macroscopical lesions. (a) Bladder exhibitingmultiple tumouBladder exhibiting a papillary neoplasm. (c) Bladder exhibititration of the muscularis propria. The lesion occupies a largepithelial abnormalities, namely cystitis cystica, von Brunn n

metaplasia’’) or was associated with cystitis cystica(‘‘cystitis glandularis’’). Nephrogenic adenoma, whichwas found comparatively rarely, usually took theform of a proliferation in the lamina propria of struc-tures similar to renal tubules, lined by cuboidal orlow-columnar epithelial cells (Fig. 2c). In all thelesions described above, epithelial cells showed little ifany atypia.

Neoplasms

The location of tumours within the bladder wall variedgreatly. In some cases (141/373) neoplasia was con¢nedto a single site, but in themajority multiple tumours oc-curred within the same bladder (Fig. 3a).The gross appearance of neoplastic lesions varied

greatly. Exophytic growths, which varied in diameterfrom a few millimeters to several centimetres, hadan attachment to the bladder wall which was eitherpedunculated or broad, and a surface which waseither smooth or branch-like (Fig. 3b). Depending on

rs, two of themcorresponding to exophytic haemangiosarcomas. (b)ng an endophytic neoplasm (transitional cell carcinoma) with in¢l-e portion of the bladder surface. In an adjacent area non-neoplasticests and urothelial hyperplasia, were also diagnosed. Scales, mm.

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Table 2Histopathological types and numbers of the bladder neo-

plasms

Details of tumours n Percentage (%)

Epithelial 539 62.0BenignPapilloma 84 9.6Adenoma 8 0.9

Papillary neoplasm of apparent lowmalignant potential (PNALMP)

55 6.3

MalignantTransitional cell carcinoma 359 41.4Adenocarcinoma 20 2.3Squamous cell carcinoma 13 1.5

Mesenchymal 331 38.0BenignHaemangioma 256 29.5Fibroma 18 2.0

MalignantHaemangioendothelioma 14 1.6Haemangiosarcoma 43 4.9

Total number of tumours identi¢ed 870 100

T. Carvalho et al.340

vascularization, these tumours were either pale or hae-morrhagic. Endophytic growths took the formof space-occupying lesions of thebladder wall, varying fromdis-crete undulations of the mucosawith moderate changeof colour, to deeply in¢ltrating growths that sometimescovered extensive areas (Fig. 3c).With few exceptions,themacroscopical appearance gave no clear indicationof histological pattern.As expected, a considerable variety of neoplastic le-

sions was observed.Tumours occurred either as a singletypewithin the samebladder (regardless of the numberof tumour masses) or as a combination of di¡erenttypes (Table1). Of these 870 tumours, 52%weremalig-nant and 42% benign, the remaining 6% beingaccounted for by epithelial papillary neoplasms ofapparently low malignant potential (PNALMP)(Table 2).Epithelial neoplasms. These were more common (62%)than mesenchymal tumours (38%) (Table 2). Of thebenign epithelial tumours, papillomas were the mostcommon (84/870); these exophytic lesions hada delicate¢brovascular stroma forming papillary fronds coveredwith transitional epithelium which was indistinguish-able from the normal urothelium, both in the numberof cell layers and in their polarity. A variant not pre-viously described in domestic animals but regularlyobserved in the present study was the inverted papillo-ma, a tumour with characteristics of a transitional cellpapilloma in which the papillary projections showedan endophytic growth pattern, with invaginations ex-tending into the stroma (Fig. 4). Areas of invertedgrowth were also frequently identi¢ed within papillo-mas and also transitional cell carcinomas.

TableDetails and numbers of bladder tumours in the

Present study

Details of tumours n %

Epithelial in pure form 191 51.2Benign 31 8.3Malignant 160 42.9Epithelial in combinationwithmesenchymal

117 31.4

Benign 19 5.1Malignant 98 26.3Mesenchymal in pure form 65 17.4Benign 48 12.9Malignant 17 4.6

Total number of bladders with tumours 373Total number of tumours identi¢ed 870

For the purpose of constructingTable 1, PNALMP (papillary neoplasms ofdicated.

Adenomas (8/870) were cauli£ower-shaped or ped-unculated and characterized by glandular structuresthat showed few if any signs of cellular atypia.PNALMP (55/ 870) were similar to the typical papil-

loma, with minimal variation in the architectural andnuclear features, yet showing increased cellular prolif-eration, exceeding six cell layers in thickness (Fig. 5).Signs of cellular atypiawere rare or non-existent.These

1present study and in three previous studies

Ozkul and Aydin

(1996)

Pamucku et al. (1976) Xu et al. (1989)

35.3% 35.2% 36.7%24% 17% NI4% 16.5% NI

25.4% 53.9% 17.5%

19% 9.3% NI4% 44.6% NI

46.6% 9.3% 45.4%43.2% 5.7% NI3.4% 3.6% NI

815 139 3541063 NI NI

apparent low malignant potential) were regarded as benign. NI, not in-

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Fig. 4. Inverted papilloma. Bovine bladder. HE. Bar,150 mm.

Fig.5. Papillary neoplasm of apparent lowmalignant potential. Bo-vine bladder. HE. Bar, 200 mm; inset bar, 20 mm.

Urinary Bladder Lesions in BEH 341

neoplasms, although sharing features common to pa-pilloma and to transitional cell carcinoma, could notcon¢dently be placed in either of these two categories.Transitional cell carcinoma (TCC) was by far the

most common lesion found in bovine urinary bladders(359/870), consisting entirely, partly or focally of ana-plastic transitional epithelium. TCCs were subclassi-¢ed according to several criteria. In terms of growthpattern, lesions were either papillary, invasive, a com-bination of both, or non-papillary and non-in¢ltrating.The degree of invasiveness was evaluated with regardto the presence or absence of neoplastic cells withinthe lamina propria, submucosa or muscularis propria,on which criterion tumours were assigned to one of thethree following categories: Ta, with no in¢ltration

(48.7%);T1, in¢ltration con¢ned to the laminapropria,including the muscularis mucosae (46%); T2, in¢ltra-tion reaching the muscularis propria (5.3%). Di¡erenthistological variants or patterns of TCCs were identi-¢ed, namely micropapillary, nested and microcystic.‘‘Micropapillary’’ referred to the formation of rami¢edbranches distributed in the tumour stroma, assuming apapillary growthpattern (Fig.6a).The‘‘nested’’variantconsisted of deep in¢ltrations into the laminapropria ofsmall rounded cell aggregates, more numerous andsmaller in size thanVon Brunn nests, showing mild nu-clear atypia (Fig. 6b).The ‘‘microcystic’’ pattern corre-sponded to transitional cell growths that formed cystswith various shapes; the epithelium lining these cystshad various numbers of cell layers (Fig. 6c). Non-papil-lary and non-in¢ltratingTCC (carcinoma in situ) wasoccasionally identi¢ed in the vicinity of other tumourlesions but never by itself (Fig. 6d).When tumoural growth followed a glandular pat-

ternwith signs of malignancy, adenocarcinomawas di-agnosed (20/870). Occasionally, these lesions showedareas of signet-ring cell proliferation (Fig.7).Squamous cell carcinomas (13/870) were character-

ized by extensive areas of squamous di¡erentiationwith keratin pearl formation. Such tumours were fre-quently large and exuberant, occupying most of thebladder surface and displaying an in¢ltrating growthpattern, frequently reaching the muscularis propria.Mesenchymal neoplasms.These showed limited variation,being predominantly of vascular origin. Haemangio-ma was diagnosed on the basis of the same criteria asthose used for the diagnosis of these lesions in other or-gans and tissues. Cavernous or capillary, this tumourwas the second most common neoplasm of bovineurinary bladder (256/870), consisting of a multitude ofvessels withwell di¡erentiated capillary-type endothe-lium, arising in well-de¢ned areas of the laminapropria. The growth pattern was either endophyticor exophytic, the latter term referring to polyps whoselamina propria was occupied by the haemangiomaitself.Haemangiosarcoma (43/870) was diagnosed in cases

in which the tumour mass was composed of blood ves-sels, those still recognizable as such being either smalland rare or large and anastomising, highly in¢ltrating,with both solid and cystic areas, and with the constantpresence of cellular atypia.Haemangioendothelioma (14/870) represented an in-

termediate category between haemangioma and hae-mangiosarcoma, consisting of some areas of solidgrowth and others in which vessel formation was ob-vious (Fig. 8). Cellular atypia was rare and in¢ltratingcapability was apparent in discrete foci.The ¢broma, which was the only non-vascular me-

senchymal tumour observed (18/870), was character-

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T. Carvalho et al.342

ized by a well-circumscribed, non-encapsulated prolif-eration of ¢broblasts, showing few if any signs of atypia,and rich in intercellular stroma (Fig. 9).The macrosco-

Fig. 6a^d. Transitional cell carcinoma of the urinary bladder. Di¡erentnested variant, and (c) microcystic. (d) Carcinoma in situ. B20 mm (d).

Fig. 7. Adenocarcinoma, signet ring variant, with invasion of themuscularis propria by the neoplastic cells. Bovine bladder.Periodic acid-Schi¡. Bar,150 mm; inset bar, 20 mm.

pical appearance was always that of small (2^4mmdiameter), well-demarcated white nodules on the blad-der surface.

histological variants were identi¢ed, namely (a) micropapillary, (b)ovine bladder. HE. Bar,150 mm (a, b, and c). Bar,100 mm; inset bar,

Fig. 8. Haemangioendothelioma, with invasion of the muscularispropria. Bovine bladder. HE. Bar, 200 mm; inset bar, 20 mm.

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Fig. 9. Fibroma. Bovine bladder. HE. Bar, 200 mm. Inset: gross ap-pearance; scale, mm.

Urinary Bladder Lesions in BEH 343

Discussion

This report illustrates the considerable histologicalvariety of urinary bladder lesions in cattle in Sa� o Mi-guel Island, where BEH is endemic.Bladder tumours associated with BEH are common

in other parts of theworld, andTable1shows the resultsof previous similar studies (Pamukcu et al., 1976; Xu,1992; Ozkul and Aydin, 1996). The results of Pamukcuet al. (1976) were similar to those described here in re-spect of epithelial versus mesenchymal tumours, butthe earlier reports did not present detailed histologicaldescriptions, making it di⁄cult to compare classi¢ca-tion criteria. Nevertheless, the types of tumour de-scribed generally resembled those of the present study,except for the non-epithelial tumours. Thus, in ourstudy, other than ¢bromas, only tumours of vascularorigin were identi¢ed, whereas ¢brosarcomas, leio-myosarcomas, rhabdomyosarcomas and round cell tu-mours were diagnosed by the three previous studies.The present study also con¢rmed the ¢ndings of othersregarding the diagnosis of haemangioendothelioma(Pamukcu et al., 1976; Ozkul and Aydin, 1996; Pires,1998), a type of vascular tumour not included inWHOclassi¢cations (Pamukcu, 1974; Meuten et al., 2004),although mentioned by Pamukcu et al. (1976). Carcino-sarcoma (Pamukcu et al.,1976; Ozkul and Aydin,1996)

was not identi¢ed in our samples. Variation in the re-sults of studies from di¡erent parts of the world maybe attributed to di¡erences in sample size, species ofbracken, and criteria used in the collection of bladdersamples at slaughter, as suggested by other authors(Xu,1992; Ozkul and Aydin,1996).The histological classi¢cation of urinary bladder le-

sions in BEH took account of diagnostic criteria com-monly used for both domestic animals and man. Allthe lesions described fell into one of three di¡erent ca-tegories, namely in£ammatory lesions, non-neoplasticepithelial abnormalities (NNEA) and neoplasms. In-£ammatory lesions, namely polypoid and follicular cy-stitis, were similar to those described in other species(Maxie, 1993). NNEA corresponded to a category ofproliferative epithelial changes of uncertain biologicalpotential, also recognized in human pathology (Mos-to¢ et al.,1999). Such lesionsmaybe part of a continuumthat culminates in true neoplasia (Amin and Young,1997; Oyasu, 2000). According to Oyasu (2000), hyper-plasia, initially £at and subsequently exophytic or en-dophytic, or both, occurs in the rat bladder afterexposure to chemical carcinogens; it may be reversibleor progressive depending on the degree of exposure. Inthe present study, NNEAwere identi¢ed in 133 of the433 bladders, and only in 26 was no coexisting neoplas-tic lesion present. In the human bladder, hyperplasia isalso frequently associated with neoplasms, the grade ofhyperplasia apparently being related to the grade of tu-mour (Amin andYoung,1997).Several NNEA described in the present study were

also considered in theWHO classi¢cation of urinarybladder tumours of domestic animals (Meuten et al.,2004) to be tumour-like lesions, with the exception ofnephrogenic adenoma. This lesion, which was rare inthe present study, has probably been interpreted di¡er-ently by other authors. Nephrogenic adenoma is a me-taplastic lesion classi¢ed by human pathologists as anepithelial abnormality (Mosto¢ et al., 1999), and hasbeen regarded as a precursor to neoplasia, due to itspropensity to recur and its morphological similarity toadenocarcinoma of the bladder (Amin and Young,1997).The neoplasms described generally accorded with

the categories devised for all animal species (Meutenet al., 2004), but there were some exceptions.Within be-nign epithelial tumours, inverted papilloma, recog-nized for the ¢rst time in human pathology by Pottsand Hirst (1963), and described in typing of humanbladder tumours (Mosto¢ et al.,1999; Eble et al., 2004),has not been recognized previously in veterinarypathology. However, in the authors’opinion, this typeof tumour at present deserves to be considered sepa-rately from papillomas; future studies may determinewhether the inverted growth pattern is associated with

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T. Carvalho et al.344

an increased risk of subsequent carcinoma develop-ment.PNLMP, identi¢ed in 6% of the bladder tumours,

represented a type of papillary neoplasm thatshared features with both papilloma and transitionalcell carcinoma; it was, however, di⁄cult to place ineither categories because the number of cell layersof the urothelium was higher than normal and epithe-lial atypia was not present. Recently, the WHOclassi¢cation for human bladder tumours (Epsteinet al., 1998; Mosto¢ et al., 1999; Eble et al., 2004) intro-duced a new category, the papillary neoplasm oflow malignant potential, similar in most aspectsto the lesions described in the bovine bladder. Studieson the grading of urothelial carcinoma have alsotaken account of this type of tumour (Cheng et al.,2000). In BEH, the absence of follow-up studiesmakes it impossible to be certain whether theseneoplasms possess low malignancy; for this reasonthey were named papillary neoplasms of apparent lowmalignant potential.With regard tomalignant epithelial tumours, noma-

jor di¡erenceswere noted fromother studies (Pamukcuet al., 1976; Xu,1992; Ozkul and Aydin,1996). Reactiveatypia was occasionally seen in bladders severely af-fected by in£ammation. Also, sporadic cases of nucleo-megaly were identi¢ed, consistent with the descriptionof cell crowding and loss of polarity typical of carcino-ma in situ (McKenney et al., 2001).These non-papillaryand non-in¢ltrating tumours, similar to those pre-viously described (Borzacchiello et al., 2001, 2003a),were not included in our analysis, as they were alwaysseen in the transitional epithelium adjacent to othertypes of tumour, and never as an isolated neoplasm.They were, therefore, interpreted as severe dysplasia,resulting from the highly aggressive tumours in the sur-roundings.Evidence suggests that the amount and duration of

bracken exposure play a crucial role in the frequency,nature and severity of BEH lesions (Shahin et al.,1998b). Hence, the development of bladder tumours inBEH is probably part of a continuous process, with aty-pia, dysplasia and carcinoma in situ corresponding todistinct successive stages of the same process, with theborderlinebetween these categories sometimes di⁄cultto establish.Although not included in theWHO classi¢cation of

urinary bladder tumours in domestic animals (Meutenet al., 2004), haemangioendothelioma has been de-scribedbyother authors in the context of BEH (Pamuk-cu et al., 1976; Ozkul and Aydin, 1996; Pires, 1998). Inhuman pathology this neoplasm is recognized in otherorgans and tissues, falling between haemangiomas andhaemangiosarcomas in terms of invasiveness and cellu-lar atypia, and hence corresponding to a low-grade

malignant vascular tumour (Calonje and Fletcher,1995).The large numbers and types of neoplasms found in

bladders collected from cattle a¡ected by BEH at Sa� oMiguel Island slaughterhouse emphasizes the need toopen the urinary bladders of all cattle from brackenfern-infested areas, even those not exhibiting clinicalsigns at slaughter. This precaution was also supportedby Sardon et al. (2005) in respect of cattle exposed tobracken fern in Spain.In BEH no association between the histological

grade of bladder lesions and their biological behaviourcan be made, as samples are obtained at slaughter andthere is, therefore, no treatmentor follow-up.Neverthe-less, there is unquestionable merit in the use of animalmodels for studying the pathogenesis of bladder neo-plasms.A future extension of the present work lies in the

identi¢cation of the various pathways for the develop-ment of each speci¢c type of neoplasm, particularly inthe light of increased understanding of the molecularmechanisms that play a role in carcinogenesis.

Acknowledgments

The authors thank ProfessorJorge Soares andDr PedroOliveira from the Department of Pathological Mor-phology of the Portuguese Institute of Oncology, Fran-cisco Gentil, Lisbon, for advice in the interpretation ofsome of the bladder lesions. Also acknowledged is DrIolanda Fernandes and Mrs Maria do RosaŁ rio Luisfor expert technical assistance, and Dr Gonc-alo Forjazfor the sampling and macroscopical photographs.Thisresearch was supported by the Foundation for ScienceandTechnology, project POCTI 34320.

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Received,March 31st, 2005

Accepted, January 27th, 2006

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