Neotrop. Helminthol., 8(2), 2014
2014 Asociación Peruana de Helmintología e Invertebrados Afines (APHIA)
ISSN: 2218-6425 impreso / ISSN: 1995-1043 on line
ORIGINAL ARTICLE / ARTÍCULO ORIGINAL
FIRST COMPARATIVE MORPHOLOGICAL STUDY OF FASCIOLA HEPATICA (LINNAEUS,
1758) FROM BRAZIL AND ARGENTINA
PRIMER ESTUDIO MORFOLÓGICO COMPARATIVO ENTRE FASCIOLA HEPATICA
(LINNAEUS, 1758) PROVENIENTE DE BRASIL Y ARGENTINA
Jéssica de Assis Santos¹, Fernanda Barbosa de Almeida da Cunha¹, Eduardo José Lopes Torres², Renata Heisler Neves²,
5 3 3,4
Daniel Daipert-Garcia¹, Jorge Bruno Malandrini , Maria Laura Pantano , Jorge Nestor Velásquez, Silvana Carnevale ,
7
Adriana Mello Garcia , José Roberto Machado-Silva² & Rosângela Rodrigues-Silva¹.
¹Laboratório de Helmintos Parasitos de Vertebrados- Fiocruz/RJ, Brazil
²Laboratório de Helmintologia Romero Lascasas Porto- Universidade do Estado do Rio de Janeiro, Brazil
3Instituto Nacional de Enfermedades Infecciosas – ANLIS “Dr. Carlos G. Malbrán”
4 5
Consejo Nacional de Investigaciones Científicas y Técnicas (CONCEIT) Faculdad de Ciências de La Salud. Universidad Nacional de Catamarca
6 7
Hospital Municipal de Infecciosas “Dr. Francisco Javier Muñiz” Departamento de Medicina Veterinária - Universidade de Lavras - Brazil
Suggested citation: Santos, JA, da Cunha, FBA, Torres, EJL, Neves, RH, Daipert-Garcia, D, Malandrini, JB, Pantano, ML,
Velásquez, JN, Carnevale, S, Garcia, AM, Machado-Silva, JR & Rodrigues-Silva, R. 2014. First comparative morphological
study of Fasciola hepatica (Linnaeus, 1758) from Brazil and Argentina. Neotropical Helminthology, vol. 8, n°2, jul-dec, pp. 393-
402.
Abstract
Keywords: Argentina - Brazil - Fasciola hepatica – light microscopy - morphological study.
Fascioliasis is an infection posing a substantial threat to public health, besides causing economic
impacts due to its increasing prevalence in recent years. It is considered endemic in Peru and Bolivia
and in Brazil's southern state of Rio Grande do Sul. Studies conducted with specimens of Fasciola
hepatica from different geographic regions have shown that the characteristics of the adult worms and
eggs vary according to host's distribution. In this study, we evaluated whether these variations also
occur in specimens obtained from cattle bred in Brazil (Cordeiro in Rio de Janeiro state and Lavras in
Minas Gerais) and Argentina (Catamarca and Salta provinces), using light microscopy and light
microscopy with differential interference contrast. The results demonstrated differences in the
specimens from each locale, mainly in the distribution and quantity of spines in the tegument. The
integrated use of various microscopic techniques is necessary to gain a better understanding of the
morphology of this parasite.
Resumen
Palabras clave: Argentina – Brazil – estudio morfológico - Fasciola hepatica – microscopía de luz.
La fasciolosis ha sido considerada una infección de gran importancia para la salud pública, además de
causar impactos en la economía debido al aumento del número de casos en los últimos años. Es
considerada endémica en Perú y Bolivia. En Brasil, la enfermedad es endémica en el estado de Río
Grande do Sul. Estudios realizados con especímenes de Fasciola hepatica de diferentes regiones
geográficas demuestran que las características de vermes adultos y huevos pueden variar de acuerdo
con la localización de origen del hospedero. En este estudio evaluamos si estas variaciones también
ocurren en especímenes obtenidos de bovinos provenientes de Brasil (Cordeiro/ Rio de Janeiro y
Lavras/ Minas Gerais) y Argentina (Catamarca y Salta), utilizando microscopia de campo claro y
microscopia de campo claro con interferencia diferencial. Los resultados demonstraron diferencias en
las muestras de cada localidad, principalmente en la distribution y cantidad de espinas en el tegumento.
El uso integrado de diversas técnicas microscópicas es necesario para obtener una mejor comprensión
de la morfología de este parásito.
393
Santos et al.
Comparative morphological of Fasciola hepatica
Fasciola hepatica (Linnaeus, 1758) is a
flatworm that causes the zoonosis fascioliasis.
Its occurrence is mainly associated with areas of
cattle and sheep breeding. These animals are its
most frequent final hosts, with freshwater snails
in nearby water bodies serving as intermediate
hosts (Bargues et al., 2012). Infection in water
buffaloes, goats, horses (Pile et al., 2001), pigs
(Araújo et al., 1995), various wild animals and
humans (Apt et al., 1993) is less frequent. The
disease, which affects the liver, is of great
economic and veterinary importance due to the
losses caused by unfitness of liver for
consumption, reduced wool, milk and meat
production, secondary infections, weight loss,
interference in fertility and even death of
animals, besides the high cost of anthelmintic
treatment (Queiroz et al., 2002). Also,
fascioliasis is one of the main food-borne
parasitic diseases in humans (Gulsen et al.,
2006).
The disease has wide global distribution (Gulsen
et al., 2006). In Europe, cases have been reported
in France, Portugal, Spain and the United
Kingdom (Esteban et al., 1998), while it has also
been reported with rising frequency in many
countries in Africa, Asia and Oceania (Kimura et
al., 1984; Şakru et al., 2011).
In South America the disease is common in
Bolivia (Fuentes, 2006), Peru (Espinosa et al.,
2010, González et al., 2011) and Argentina
(Mera y Sierra et al., 2009). In Argentina
specifically, human cases of fascioliasis have
been reported in the provinces of Buenos Aires,
Córdoba, Catamarca, San Luis and Mendoza
(Mera y Sierra et al., 2011).
In Brazil, cases have been confirmed in animals
and humans in the states of Rio Grande do Sul,
Santa Catarina, Paraná, São Paulo, Rio de
Janeiro and Minas Gerais (Gomes et al., 2002).
In Rio Grande do Sul, the disease is considered
endemic (Cunha et al., 2007). A study by Dutra
et al. (2010) confirmed that this zoonosis occurs
most often in southern Brazil, although there
have also been cases in the southeastern region,
in Espírito Santo and Rio de Janeiro (Alves et al.,
2011, Bennema et al., 2014).
While molecular techniques have been applied
successfully for genotypic characterization (Ai
et al., 2011), phenotypic analysis is also
important in systematic studies of the genus
Fasciola. Studies applying light microscopy
have demonstrated morphological variations
between adult specimens from Bolivia, Spain
and Corsica (Valero et al., 2005; Periago et al.,
2006) and eggs of F. hepatica from Bolivia
(Valero et al., 2005), Vietnam, Georgia and
Egypt (Valero et al., 2009). On the other hand,
light microscopy with differential interference
contrast (DIC), or Nomarski microscopy, has
been applied to obtain detailed knowledge of the
morphological structures of trematodes and
especially for taxonomic purposes (Moravec et
al., 2002; Gomes et al., 2002; Singh et al., 2014).
However, investigations of possible
phenotypical variations and techniques that can
identify new morphological traits of F. hepatica
have not been comprehensively employed in
Brazil. This article compares the phenotypical
traits of isolates of F. hepatica from different
regions of Brazil and Argentina by means of
light microscopy with and without DIC.
The adult specimens were collected from
naturally infected cattle at abattoirs in the
Brazilian municipalities of Cordeiro, Rio de
Janeiro (22°01'43”S - 42°21'33”W) and Lavras,
Minas Gerais (21°14'S - 45°00'W) and in the
Argentine provinces of Salta (24°47'00”S -
65°25'00”W) and Catamarca (29°16'00”S –
65°3'34”W).
The study method applied was based on the
protocol proposed by Valero et al. (2005). The
adult specimens were fixed in 70% ethanol,
pressed between glass slides to facilitate
visualization of the structures and stained with
alcoholic chlorhydric carmine (Langeron,
1949). Immediately thereafter, the samples were
dehydrated in an ascending alcohol series
INTRODUCTION
MATERIALS AND METHODS
394
Figure 1. Morphological structures of Fasciola hepatica from cattle in the municipality of Cordeiro, RJ, Brazil revealed by light
microscopy. (A) Cephalic cone. OS, oral sucker; I, intestinal ceca; PH, pharynx; VS, ventral sucker; spines (arrows).
Magnification 4x. (B) Uterus filled with eggs (arrows). Magnification 10x.
Neotrop. Helminthol., 8(2), 2014
followed by clarification with a 1:1 solution of
methyl salicylate and Canada balsam. The
material was then mounted on slides, which
w e r e f i l l e d w i t h C a n a d a b a l s a m .
Photomicrographs were obtained with a light
microscope (Olympus BX51). For the same
samples, photomicrographs were also taken
using differential interference contrast (DIC), to
enable observing the spines on the tegument
more clearly. The study was conducted with
material collected from slaughtered cattle. So it
is not applicable to an assessment of the ethics
committee.
Light microscopy
One hundred and two specimens were analyzed.
Forty-six specimens from Cordeiro- Rio de
Janeiro, eighteen specimens from Lavras- Minas
Gerais, twenty from Catamarca and fifteen from
Salta, Argentina. Regardless of geographic
origin, all the specimens presented a dorso-
ventrally flattened, leaf-shaped body, with a
very evident cephalic cone located in the
anterior part of the body (Figure 1a).
Besides these features, a rounded oral sucker
was observed at the apex of the cone, followed
by a muscular pharynx and stomach. The
esophagus bifurcated into two highly dendritic
intestinal ceca, from which secondary branches
emerged (Figure 2a). The intestinal ceca
extended to the anterior end of the body (Figure
2b). The ventral sucker was located in the middle
region of the body, near the reproductive
apparatus, and was larger than the oral sucker.
The male reproductive system contained
branched testes located one after the other,
without reaching the hind region or the sides of
the body, having the vitelline glands as limits. A
well-formed cirrus pouch was observed near the
ventral sucker. The female reproductive system
was formed by a branched ovary, oviduct and
uterus, which was filled with eggs in the
specimens analyzed (Figure 2c – 1b). The
vitelline glands were positioned on the side of
the body and were highly branched, extending
from the region of the ventral sucker to the
posterior body end. The Mehlis gland, located
near the ovary in the middle part of the body, was
easily observed.
All the specimens from Cordeiro, Catamarca
and Salta presented tegument covered with
spines (Figures 1b; 2b; 3b). These spines were
pointed toward the hind part of the body. All the
specimens from Lavras, it was not possible to
see spines on the tegument (Figure 4).
RESULTS
395
Santos et al.
Comparative morphological of Fasciola hepatica
Figure 2. Morphological structures of Fasciola hepatica from cattle in the province of Catamarca, Argentina revealed by light
microscopy. (A) cephalic cone. OS, oral sucker; I, intestinal ceca; PH, pharynx; C, cirrus; VS, ventral sucker. Magnification 4x.
(B) Uterus filled with eggs (arrows). (C) Detail of the cephalic cone showing the spines (arrows); I, intestinal ceca. Magnification
10x.
396
Li g ht microsco py with differe n tial
interference contrast
This technique was applied to observe 23
specimens. Eight specimens from Cordeiro, Rio
de Janeiro, seven specimens from Lavras, Minas
Gerais, four specimens from Catamarca and four
specimens from Salta, Argentina. It was possible
to visualize spines on the tegument of specimens
from all the collection sites. On the specimens
from Cordeiro and Salta (Figures 5 and 6), these
spines were in larger quantities, with a different
pattern of distribution, but with the same aspect,
than the specimens from Catamarca and Lavras,
on which very few spines were observed
(Figures 7 and 8).
Neotrop. Helminthol., 8(2), 2014
Figure 3. Morphological structures of Fasciola hepatica from cattle in the province of Salta, Argentina revealed by light
microscopy. (A) cephalic cone. OS, oral sucker; I, intestinal ceca; PH, pharynx; C, cirrus; VS, ventral sucker; spines at the edge of
the tegument (arrows). Magnification 4x. (B) Detail of the cephalic cone showing the spines (arrows). Magnification 10x.
Figure 4. Morphological structures of Fasciola hepatica from cattle in the municipality of Lavras, MG, Brazil revealed by light
microscopy. (A) cephalic cone. OS, oral sucker; I, intestinal ceca; PH, pharynx; E, esophagus; C, cirrus pouch. Magnification 4x.
(B) Uterus filled with eggs (arrows); C, Cirrus; I, intestinal ceca; VS, ventral sucker. Magnification 10x.
Figure 5. Morphological structures of Fasciola hepatica from cattle in the municipality of Cordeiro, RJ, Brazil revealed by light
microscopy with differential interference contrast. Final portion of the cephalic cone, showing the spines (arrows) and I, intestinal
ceca.
397
Santos et al.
Comparative morphological of Fasciola hepatica
Figure 6. Morphological structures of Fasciola hepatica from cattle in the province of Salta, Argentina revealed by light
microscopy with differential interference contrast. Final portion of the cephalic cone, showing the spines (arrows); OS, oral
sucker.
Figure 7. Morphological structures of Fasciola hepatica from cattle in the province of Catamarca, Argentina revealed by light
microscopy with differential interference contrast. Final portion of the cephalic cone, showing the spines (arrows).
Figure 8. Morphological structures of Fasciola hepatica from cattle in the municipality of Lavras, MG, Brazil revealed by light
microscopy with differential interference contrast. (A, B) with differential interference contrast. Final portion of the cephalic
cone, showing the spines (arrows); and I, intestinal ceca.
398
Neotrop. Helminthol., 8(2), 2014
Light microscopy has been widely used to study
the morphology of different developmental
phases of trematodes, to establish the taxonomy
or provide more information on their structures.
Over the past decade, this technique has made
important contributions to the study of adult
worms and eggs of F. hepatica (Valero et al.,
2005; Periago et al., 2008; Valero et al., 2012).
The appearance of the eggs in this study did not
differ from other descriptions in the literature
(Hussein et al., 2010a,b). We found
morphological similarities of the adult
specimens from Cordeiro, Salta and Catamarca,
which did not differ from reports on Brazilian
specimens from the city of Porto Alegre
(Marques, 2002), Goiás state (Araújo et al.,
1995), and the cities of Careaçu and Itajubá
(Oliveira, 2008). Even populations from
geographic areas that are far apart (Spain and
Bolivia) can present only small allometric
differences (Valero et al., 1999).
By means of light microscopy it was not possible
to observe the presence of spines on the
tegument of the specimens from Lavras,
although the other structures analyzed of these
specimens were similar to those of specimens
from the other geographic regions. The presence
of spines on the tegument helps the helminth's
fixation in its habitat. Besides this, the tegument
is the region of interaction between the parasite
and host, where the parasite both incorporates its
nutrients (Oliveira, 2008) and develops
strategies to overcome the host's immune system
(Moreau & Chauvin, 2010). It is speculated that
adult parasites have limited interaction with the
host, but further research is necessary to
demonstrate whether this is a phenotypic trait.
The bright field microscope with differential
interference contrast is widely used in studies
helminths, especially nematode. According to
Szmygiel et al. (2014).The technique can be
used to visualize structures that are not possible
with scanning electron microscopy,
demonstrating the need for integrated use of
such microscopy techniques (Bennet, 1975).
Despite the many advantages of light
microscopy in helminthology studies, the
characterization of certain morphological
details requires the use of other techniques, such
as differential interference contrast (Singh et al.,
2014; Szmygiel et al., 2014). With this technique
was possible visualize the difference in the
distribution of spines in the tegument of
Fasciola hepatica. To the best of our
knowledge, this paper is the first to report the use
of this technique on F. hepatica. With the
integrated use of the two techniques, it was
possible to observe spines also on the specimens
from Lavras, evidencing the need for integration
of microscopic techniques to obtain reliable
results. While most of the specimens of F.
h e p a t i c a exa m i n ed h e r e p r e sented
morphological similarity when analyzed by light
microscopy alone, the integrated use of various
microscopic techniques is necessary to gain a
better understanding of the morphology of this
parasite.
DISCUSSION
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Received September 23, 2014.
Accepted Novmeber 29, 2014.
