Neotrop. Helminthol., 5(1), 2011

2011 Asociación Peruana de Helmintología e Invertebrados Afines (APHIA)

ISSN: 2218-6425 impreso / ISSN: 1995-1043 on line

Resumen

Abstract

ORIGINAL ARTICLE / ARTÍCULO ORIGINAL

LIVER HISTOPATHOLOGY OF ECHINOCOCCUS GRANULOSUS INFECTION

IN SUS SCROFA FROM BRAZIL

HISTOPATOLOGÍA DEL HÍGADO CAUSADA POR LA INFECCIÓN POR

ECHINOCOCCUS GRANULOSUS EN SUS SCROFA PROCEDENTE DE BRASIL

1,3 1 1 1, 2

Fernanda Almeida B , Magda Oliveira S , Renata Neves H , Jessica Santos A Albanita Oliveira V , José Roberto Machado-

3 1,*

Silva ,& Rosângela Rodrigues-Silva

1 Laboratório de Helmintos Parasitos de Vertebrados, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Avenida Brasil 4365,

Manguinhos, Rio de Janeiro, 21040-900, RJ, Brasil.

2 Disciplina de Anatomia Patológica, Departamento de Patologia e Laboratórios, Faculdade de Ciências Médicas,

Universidade do Estado do Rio de Janeiro (UERJ), Rio de Janeiro, Brasil.

3 Laboratório Romero Lascasas Porto, Departamento de Microbiologia, Imunologia e Parasitologia, Faculdade de Ciências

Médicas, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Rua Prof. Manoel de Abreu 444/5 andar, Vila Isabel, Rio

de Janeiro, 20511-070, Brasil.

Suggested citation: Almeida, B.F., Oliveira, S.M., Neves, H.R., Santos, A,J., Oliveira, V.A., Machado-Silva, J.R. & Rodriguez-Silva, R. 2011.

Liver histopathology of Echinococcus granulosus infection in Sus scrofa from Brazil. Neotropical Helminthology, vol. 5, n° 1, pp.24 -30.

Museum specimens represent a valuable resource for helminthological research. We examined

formalin-fixed livers preserved for periods in excess of 50 years from naturally infected pigs in

archival material from the Helminthological Collection of the Oswaldo Cruz Institute (CHIOC),

Oswaldo Cruz Foundation (FIOCRUZ). Microscopic examination revealed evidence of parasites in

unilocular cystic structures containing a bladder worm vesicle lined with a thin laminated membrane.

The inner germinal layer contained parasite debris and a proteinaceous hydatid-like fluid. The hepatic

parenchyma showed a dense inflammatory infiltrate near the cyst, thick-walled arterial vessels, and

occasionally necrosis. The adjacent hepatic parenchyma showed inter- and intra-lobular thin fibrosis,

inflammatory cell infiltration and occasionally necrosis. Other histo-pathological sections showed a

thick laminated wall cystic structure with brood capsules in the germinal layer containing small round

projections with central internal vesicles on the inner surface, suggesting the presence of protoscolices.

Given that Echinococcus multilocularis does not occur in Brazil, our histological findings were

identified as Echinococcus granulosus in origin. In conclusion, the protocol presented in this study

highlights the possibility of achieving remarkably high quality histological results in archival

formalin-fixed and paraffin-embedded tissues retrieved from museum specimens that have been

preserved for many years.

Key words: Brazil - Echinococcus granulosus - histopathology - liver - Sus scrofa.

Los especímenes de museo representan un recurso valioso para la investigación helmintológica.

Hemos examinado los hígados fijados en formalina conservados por períodos de más de 50 años a partir

de cerdos infectados naturalmente en el material de archivo de la Colección Helmintológica del

Instituto Oswaldo Cruz (CHIOC), Fundación Oswaldo Cruz (FIOCRUZ). El examen microscópico

reveló evidencia de parásitos en estructuras quísticas uniloculares que contienen una vesícula de la

membrana del parásito recubierto con una lámina delgada. La capa interna germinal contiene los restos

del parásito y un líquido proteíco como hidatídico. El parénquima hepático mostró un denso infiltrado

inflamatorio cerca del quiste, los vasos arteriales de paredes gruesas y necrosis de vez en cuando. El

parénquima hepático adyacente mostró fibrosis delgada inter e intralobular, la infiltración de células

inflamatorias y necrosis de vez en cuando. Otros cortes histológicos mostraron una estructura de pared

gruesa laminada quística con cápsulas de cría en la capa germinal que contienen proyecciones pequeñas

y redondas con el centro de las vesículas internas en la superficie interna, lo que sugiere la presencia de

protoescólices. Teniendo en cuenta que Echinococcus multilocularis no ocurre en Brasil, nuestros

hallazgos histológicos fueron identificados como Echinococcus granulosus de origen. En conclusión,

el protocolo que se presenta en este estudio pone de relieve la posibilidad de lograr resultados

histológicos de muy alta calidad en archivos fijados en formalina y parafina de tejidos embebidos

recuperados de especímenes de museo que se han conservado durante muchos años.

Palabras clave: Brasil - Echinococcus granulosus - hígado - histopatología - Sus scrofa.

INTRODUCTION

Pig-rearing practices expose pigs to parasitic

infection, mainly, those of tapeworm origin.

Taeniid tapeworms in their larval stage remain a

significant medical and veterinary public health

concern (Thompson, 2008) and cause significant

losses to livestock and human productivity

(Jenkins et al., 2005; Eddi et al., 2006; Scala et al.,

2006). Some species are sustained by a predator-

prey cycle in which carnivore definitive hosts

harbor adult worms in the small intestine, whereas

herbivore intermediate hosts develop the

metacestode stage in organs (Thompson &

McManus, 2002). In South America, cystic

echinococcosis disease caused by Echinococcus

granulosus (Batsch, 1786) and characterized by

unilocular hydatid cyst occurs in the sheep-rearing

rural areas of southern Brazil, Uruguay, Argentina,

Chile and Bolivia (Kamenetzky et al., 2002).

Previous studies on the helminth faunae of Sus

scrofa (Linnaeus, 1758) raised in outdoor

conditions have reported Taenia hydatigena

(Pallas, 1766) larva (Cysticercus tenuicollis)

(Solaymani-Mohammadi et al., 2003), Cysticercus

tenuicollis and Cysticercus cellulosae (Järvis et al.,

2007). Epidemiological studies have revealed C.

cellulosae (Phiri et al., 2003) and E. granulosus

hydatidosis in swine from home slaughtering

(Bružinskaite et al., 2009) and slaughterhouses

(Lidetul & Hutchinson, 2007; Acosta-Jamett et al.,

2010). Furthermore, molecular studies highlight

the occurrence of host-adapted E. granulosus

strains in infected pigs (Kamenetzky et al., 2002;

Breyer et al., 2004; Daniel-Mwambete et al., 2004;

Haag et al., 2004; Cruz-Reyes et al., 2007;

Badaraco et al., 2008; Schneider et al., 2010). A

few reports have been published on parasite

histopathology in pigs: unilocular hydatid cysts

were distributed in the liver and lungs (Lidetul &

Hutchinson, 2007) or in the thoracic and abdominal

cavities (Martin-Hernando et al., 2008).

Histopathological examination showed a typical

laminated membrane (Lidetul & Hutchinson,

2007) and a fibrous capsule composed of

collagenous connective tissue with sporadic

calcification in mature cysts, whereas immature

cysts were surrounded by inflammatory infiltrate

(Martin-Hernando et al., 2008). Experimentally E.

multilocularis (Leuckart, 1863) -infected pigs

showed the largest lesions (3–8 mm in diameter),

pronounced central necrosis and dystrophic

calcifications. In contrast, most of the smallest

lesions (usually <1.5 mm in diameter) had small

compact fibrotic areas and blurred borders with

obvious fibrous infiltrations into interlobular

tissues (Deplazes et al., 2005). A few reports have

been published on parasite histopathology in

European and Australian pigs (Lidetul &

Hutchinson, 2007). Although molecular studies

have been performed for taxonomic analyses, there

is no information on the histopathology of E.

granulosus in Brazilian pigs (S. scrofa). We

examined formalin-fixed livers from naturally

infected pigs in archival material from Brazil.

Museum specimens are a valuable resource for

helminthological research (Santos & Moravec,

2009).

MATERIALS AND METHODS

We studied formalin-fixed livers from two S. scrofa

he ld in the sta te of Rio de Janeiro

(22°54'S43°10W), Brazil and deposited in the

Helminthological Collection of the Oswaldo Cruz

Institute (CHIOC) by Travassos (Fig. 1). Following

paraffin embedding, 5 µm thick sections were

Histopathology of Echinococcus granulosus in Sus scrofa Almeida et al.

stained with conventional hematoxylin and eosin

and examined. Histopathological analysis and

photomicrography were obtained under a

conventional Eclipse E200 Nikon light

microscope.

RESULTS

Microscopic examination revealed evidence of

parasites in unilocular cystic structures. The

structures contained a bladder worm vesicle lined

with a thin-laminated membrane and an inner

germinal layer with parasite debris and a

proteinaceous hydatid-like fluid (Fig. 2a-b). Other

histological sections showed a thick-laminated

wall cystic structure with brood capsules (germinal

layer) containing small round projections with

central internal vesicles on the inner surface,

suggesting the presence of protoscolices and their

hooks (Fig. 2b-c).

The cyst wall was attached to the nontrabeculae

hepatic parenchyma, showing a dense

inflammatory infiltrate nearest to the cyst (Fig. 2d-

3a), thick-walled arterial vessels and occasionally

necrosis, which is consistent with vasculitis (Fig.

3b-d). The adjacent hepatic parenchyma showed

inter- and intralobular thin fibrosis, consistent with

regenerative nodules. Both the septa and bile

canaliculi showed perivascular inflammatory

infiltrate, whereas the fibrosis surrounding the cyst

presented arterial vessels with acidophilic

necrosis. The histological findings were identified

as Echinococcus in origin.

DISCUSSION

The liver is the predicted site for metacestodes of

some species of tapeworms in wild boar (Oberg &

Valenzuela, 1977; Pfister et al., 1993; Aluja et al.,

1998; Blažek et al., 1985; de-la-Muela et al., 2001;

Phiri et al., 2003; Solaymani-Mohammadi et al.,

2003; Rodrìguez et al., 2006; Järvis et al., 2007;

Lidetul & Hutchinson, 2007; Martin-Hernando et

al., 2008; Bružinskaite et al., 2009).

Although cystic echinococcosis disease is a

significant veterinary public health concern in

southern Brazil (de La Rue et al., 2006; Moro &

Schantz, 2009), records of swine infection are

limited.

In this study, we provided evidence of

Echinococcus infection in S. scrofa by means of

liver histopathology. To this end, we visited

CHIOC and identified formalin-fixed liver hydatid

cysts. Furthermore, records demonstrated that the

liver was isolated from S. scrofa in Santa Cruz (Rio

de Janeiro). This finding raises the following

question: given that Rio de Janeiro is not known to

be an endemic area for E. granulosus, what was the

origin of the infected pigs? According to historical

records, Santa Cruz harbored an important abattoir

in the last century, providing meat for consumption

in Rio de Janeiro City (Ecomuseu, 2010). In

addition, an early historical report highlighted the

presence of Echinococcifer echinococcus in the

livers of swine derived from other regions

(Travassos & Silva, 1915). At that time, however, a

characterization of parasite histopathology was not

performed. Animal trade represents a potential

source of zoonotic pathogens, as exemplified by E.

granulosus (Haag et al., 2004; Pavlin et al., 2009).

Previous studies also reported the occurrence of

hydatid cysts in pigs from abattoirs in Spain

(Daniel-Mwambete et al., 2004), Australia (Lidetul

& Hutchinson, 2007) and Chile (Acosta-Jamett et

al., 2010).

Paraffin-embedded archival material is a valuable

resource for cysticercosis (Ogunremi et al., 2004)

and hydatidosis retrospective studies (Schneider et

al., 2008). Species of Echinococcus are

characterized by a hydatid type of bladder worm

(Pfister et al., 1993; Lidetul & Hutchinson, 2007;

Bružinskaite et al., 2009). In this study, histological

examination revealed a laminated layer that gave

rise to brood capsules and protoscolices consistent

with Echinococcus infection (Lidetul &

Hutchinson, 2007; Martin-Hernando et al., 2008).

Studies demonstrate that brood capsules are not

observed in unfavorable hosts (Pfister et al., 1993);

therefore, pigs may be considered permissive hosts

for E. granulosus. The adjacent hepatic

parenchyma showed inter- and intralobular thin

fibrosis, inflammatory cell infiltration and

occasionally necrosis. The pathological changes

observed were similar to those described in wild

boar naturally infected with E. multilocularis

(Boucher et al., 2005). Given that E. multilocularis

does not occur in Brazil, our histological finding

confirmed the diagnosis of E. granulosus.

Neotrop. Helminthol., 5(1), 2011

The quality of histological material may be

affected by many factors including the age of the

specimen, type and length of fixation/preservation

and storage conditions. The protocol presented in

this study highlights the possibility of achieving

remarkably high quality histological results in

archival formalin-fixed and paraffin-embedded

tissues retrieved from museum specimens that

have been preserved for many years. In conclusion,

our study histologically demonstrated the

occurrence of E. granulosus in swine and also

confirms that specimens in museums represent a

valuable resource for helminthological research.

ACKNOWLEDGEMENTS

The authors would like to thank Rodrigo Méxas

from the Laboratório de Produção e Processamento

de Imagem Científica, IOC - Fiocruz for preparing

the images. Figure 1. Card files of the Helminthological Collection of the

Oswaldo Cruz Institute (CHIOC).

Figure 2. Histopathological

aspects of hydatic cysts in

natural infection of Sus

scrofa with Echinococcus

granulosus. a Unilocular

cystic structure containing a

bladder worm vesicle lined

by a t h in lam i nat e d

membrane and an inner

germinal layer with parasite

debris (arrow) and a

proteinaceous hydatid-like

fluid, bar 0.02 mm; b Brood

capsules (germinal layer)

containing protoscolices,

b a r 0 . 0 1 m m ; c

Protoscolices and their

hooks (arrowhead), bar 0.05

mm; d Cyst wall (*) in

h e p a t i c p a r e n c h y m a

s h o w i n g a d e n s e

inflammatory infiltrate

nearest to the cyst, bar 0.05

mm.

Almeida et al.

Histopathology of Echinococcus granulosus in Sus scrofa

Figure 3. Histopathological aspects of hepatic tissues in natural infection of Sus scrofa with Echinococcus

granulosus. a Hepatic parenchyma showing a dense inflammatory infiltrate in interlobular and intralobular septa

with hepatocyte necrosis, bar 0.01 mm; b Cyst wall exhibiting on its inner surface wall vessels permeated by

inflammatory cells and necrosis, bar 0.01 mm; c Detail of the cyst wall and necrotizing vasculitis (arrow), bar 0.05

mm; d detail of necrotizing vasculitis, bar 0.05 mm.

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*Author for Correspondence /Autor para correspondencia:

Rosângela Rodrigues-Silva

Laboratório de Helmintos Parasitos de Vertebrados,

Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Avenida

Brasil 4365, Manguinhos, Rio de Janeiro, 21040-900, RJ,

Brasil. Phone: +55-021-25621485. Fax: +55-021-

25621511.

E-mail/correo electrónico:

rsilva@ioc.fiocruz.br

Neotrop. Helminthol., 5(1), 2011