Gall bladder and extrahepatic bile duct changes in

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Acta Tropica 83 (2002) 29 – 36
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           Gall bladder and extrahepatic bile duct changes in
                 Opisthorchis 6i6errini-infected hamsters
                                  Banchob Sripa a,*, Sasithorn Kaewkes b
           a
               Department of Pathology, Di6ision of Experimental Pathology, Faculty of Medicine, Khon Kaen Uni6ersity,
                                                     Khon Kaen 40002, Thailand
                b
                  Department of Parasitology, Faculty of Medicine, Khon Kaen Uni6ersity, Khon Kaen 40002, Thailand
                  Received 20 October 2001; received in revised form 28 January 2002; accepted 12 February 2002

Abstract

   Opisthorchis 6i6errini infection is associated with several hepatobiliary diseases, but few reports have described
extrahepatic lesions in opisthorchiasis. We therefore sequentially investigated histological changes of the gall bladder
and extrahepatic bile duct in hamsters infected with 25 (group 1), 50 (group 2) and 100 (group 3) metacercariae for
up to 180 days. Acute inflammatory reactions, including congestion, neutrophil and eosinophil infiltration, occurred
in the gall bladder as early as day 7 of groups 2 and 3 and on day 14 in group 1; the extrahepatic bile ducts exhibited
the changes on day 3 post-infection (p.i.). Mononuclear cell infiltration, mucus hypersecretion and fibrosis were
gradually observed thereafter. Active inflammation reached a plateau at approximately 60 days in all infected groups.
The well-established chronic histological changes of the gall bladder and extrahepatic bile duct were fibrosis and
mononuclear cell infiltration with lymphoid aggregation and, additionally, ductal dilatation for the latter. Overall, the
pathological changes in the extrahepatic bile duct were more severe than those in the gall bladder for the same dose
and period of infection. The results demonstrate that pathological changes in the gall bladder and extrahepatic bile
duct do occur in O. 6i6errini infection and may be extrapolated to human infection. © 2002 Elsevier Science B.V. All
rights reserved.

Keywords: Opisthorchiasis; Opisthorchis 6i6errini; Gall bladder; Extrahepatic bile duct; Pathology; Hamsters

1. Introduction                                                      east Asia (IARC, 1994). Thailand is an endemic
                                                                     area for opisthorchiasis, with an estimated 13
   Liver fluke infection caused by Opisthorchis                      million people infected by O. 6i6errini (calculated
6i6errini, O. felineus and Clonorchis sinensis re-                   from 21.5% prevalence of the population) (Jong-
mains a major public health problem in many                          suksuntigul and Imsomboon, 1998). The infection
parts of Eastern Europe, the Far East and South-                     is associated with a number of hepatobiliary dis-
                                                                     eases, including cholangitis, obstructive jaundice,
  * Corresponding author. Tel.: +66-43-34-8388; fax: + 66-
                                                                     hepatomegaly, cholecystitis and cholelithiasis
43-34-8375.                                                          (Harinasuta et al., 1984). Moreover, both experi-
  E-mail address: banchob@mail.kku.ac.th (B. Sripa).                 mental and epidemiological evidence strongly im-

0001-706X/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.
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30                               B. Sripa, S. Kaewkes / Acta Tropica 83 (2002) 29–36

plicate the liver fluke infection in the etiology of’        ent intensities of infection. This is the first sequen-
cholangiocarcinoma— bile duct cancer (Thamavit               tial study that describes the histologic changes of
et al., 1978; IARC, 1994; Vatanasapt et al., 1999).          the gall bladder and extrahepatic bile duct caused
   Various aspects of the pathological conse-                by O. 6i6errini infection.
quences of O. 6i6errini infection have been studied
both in man and experimental animal models.
However, most reports describe only intrahepatic             2. Materials and methods
lesions associated with the infection. These in-
clude epithelial desquamation, epithelial hyper-             2.1. Parasite
plasia, goblet cell metaplasia, adenomatous
hyperplasia and periductal fibrosis (Bhamarapra-                Metacercariae were collected from cyprinoid
vati et al., 1978; Riganti et al., 1989). Accompany-         fish obtained from a fresh water reservoir in an
ing abnormalities in the gall bladder and                    endemic area of Khon Kaen province, Thailand.
extrahepatic bile duct have received little atten-           After removing the internal organs, the muscle,
tion. Dhiensiri et al. (1984) first described gall           fin and tail of the fish, were chopped into pieces
bladder abnormalities observed by ultrasound in              and digested with 0.7 N HCl–pepsin solution for
patients with O. 6i6errini infection. Community-             2 h at 37 °C. The digestate was serially filtered
based studies in Northeast Thailand, using ultra-            and washed several times with normal saline in a
sonography and cholecystography, demonstrated                sediment jar. The metacercariae of O. 6i6errini
a dramatic increase in the frequency and severity            were identified and collected under a dissecting
of gall bladder disease— specifically wall irregu-           microscope.
larity, enlargement, bile sludge, and poor function
among apparently healthy individuals with mod-               2.2. Animals and experimental design
erate and heavy O. 6i6errini infection (Haswell-
Elkins et al., 1991; Mairiang et al., 1992; Elkins et           Two hundred male Syrian golden hamsters,
al., 1996).                                                  aged 6–8 weeks, from the Animal Unit, Faculty
   Following anthelmintic treatment, many of the             of Medicine, Khon Kaen University, were used in
gall bladder abnormalities were eliminated, as in-           the experiments. The hamsters were divided into
dicated by the reduction of the length and re-               four groups of 50. Groups 1, 2 and 3 were in-
gained contractility (Mairiang et al., 1993). While          fected with 25, 50 and 100 metacercariae by intra-
these studies show clearly that O. 6i6errini is              gastric intubation, respectively. Group 4, a
closely associated with gall bladder abnormalities,          control group, was fed normal saline solution.
the pathology and pathogenesis of the gall blad-             The animals were kept five per cage, housed under
der disease remain unclear. In addition, the extra-          conventional conditions, fed a stock diet (C.P.
hepatic bile duct, which is also a habitat of the            Ltd., Thailand) and water ad libitum. The mainte-
liver fluke, has never been reported. Since it is not        nance and care of the experimental animals com-
possible to obtain asymptomatic gall bladders and            plied with the National Experimental Animal
extrahepatic bile ducts from infected people, we             Center guidelines for the humane use of labora-
undertook a histopathological study of the gall              tory animals. Five animals from each group were
bladder and extrahepatic bile duct in a hamster              sacrificed on days l, 3, 7, 14, 30, 60, 90, 120, 150
model infected with O. 6i6errini. This animal                and 180 post-infection (p.i.).
model is a suitable candidate for O. 6i6errini
infection and has been widely used in the investi-           2.3. Pathological study
gation of the infection including its pathology and
carcinogenesis (Wykoff and Ariyaprakai, 1966;                  Hamsters were weighed and anaesthetized with
Bhamarapravati et al., 1978; Thamavit et al.,                ether. Blood was drawn from the heart. The gall
1978). A dose-related change was designed to                 bladder and extrahepatic bile duct were examined
elucidate what may happen in humans with differ-             for gross abnormalities and then carefully mi-
B. Sripa, S. Kaewkes / Acta Tropica 83 (2002) 29–36                            31

crodissected. The dissected specimens were imme-           However, quantification or semi-quantification of
diately placed in Carnoy’s solution and fixed              the opacity of the gall bladder wall was difficult to
overnight at 4 °C. The gall bladders and extra-            measure and depended on the stage of its contrac-
hepatic bile ducts were processed separately               tion. In chronic infection, particularly from day
through a series of absolute ethanol and xylene,           90 onwards, the most striking lesions were ob-
and embedded in paraffin. Sections were cut and            served in the extrahepatic bile duct. These in-
routinely stained with H&E, alcian blue– PAS and           cluded ductal dilatation, increased opacity
Masson’s trichrome for a histopathological study           (thickened wall) and periductal nodule formation.
(Luna, 1968). The liver was also excised, weighed          Qualitatively, the severity of these changes was
and kept in normal saline for worm counting.               more pronounced in groups 2 and 3 than in group
                                                           1. Opisthorchis worms were commonly seen in the
2.4. Determination worm counts                             gall bladders and extrahepatic bile ducts, espe-
                                                           cially in group 3. Typical pictures of the gross
  Worm counts were determined by direct teasing            lesions of the gall bladder and extrahepatic bile
of the liver, and from the number of flukes seen in        duct in the control and infected groups are shown
the histologic sections of the gall bladder and            in Fig. 1a–d.
extrahepatic bile duct.
                                                           3.3. Histological findings

3. Results                                                    Histologically, the gall bladder of the control
                                                           hamster consists of three layers: the mucosa which
3.1. Parasitological data                                  includes surface columnar epithelia and lamina
                                                           propria, the muscle and the thin serosa. The
   The average worm recovery rate in the ham-              extrahepatic bile duct also contains three layers
sters infected with O. 6i6errini in groups 1, 2 and        but with a thin lamina propria. A few
3 from day 30 to 180 p.i. was 50, 55 and 47%,              lymphocytes were seen in the lamina propria of
respectively. Details has been published previously        the gall bladder. No significant change was ob-
(Sripa and Kaewkes, 2000a).                                served in either the gall bladder or extrahepatic
                                                           bile duct of the hamsters in the control group,
3.2. Gross findings                                        other than a mild mononuclear cell infiltration at
                                                           the cystic duct junction of the gall bladder in
   The gall bladder in the control group (group 4          some animals.
was uninfected) showed a transparent wall with                During the 1st week of infection, no micro-
varying amounts of bile. The extrahepatic bile             scopic changes in the gall bladders were observed
duct, which includes the main common hepatic               in any of the infected groups. Mild edematous
ducts and the common bile duct, was small and              villi, congestion and some neutrophil infiltration
transparent. Its total length was approximately 1          were first observed on day 7 p.i. in some hamsters
cm from the duodenal junction. The gall bladder            in groups 2 and 3 and on day 14 p.i. in group 1.
and extrahepatic bile duct in this group showed            A number of eosinophils were also observed.
no gross abnormality during the 180 days of the            Mononuclear        cell    infiltration   including
study.                                                     macrophages, lymphocytes and plasma cells, in-
   The gross appearance of the gall bladders and           creased gradually until the end of the experiment,
extrahepatic bile ducts in the infected groups 1, 2        especially in groups 2 and 3. Mild fibrosis of the
and 3 was unremarkable during the first 14 days            gall bladder wall was seen from day 30 onwards.
p.i. From day 30 on, the gall bladder and extra-           Epithelial hyperplasia and mucus secretion were
hepatic bile duct wall were slightly opaque, espe-         not as prominent in infected gall bladders as in
cially in group 3. The severity of the opacity             the extrahepatic bile ducts. The mucosa in contact
increased gradually with the duration of infection.        with the flukes showed signs of atrophy and gob-
32                                    B. Sripa, S. Kaewkes / Acta Tropica 83 (2002) 29–36

let cell metaplasia. These pathological changes                     standing this, epithelial desquamation was not
were even more pronounced in the extrahepatic                       seen in any of the groups studied. The histological
bile ducts during the same period. Epithelial dis-                  changes gradually worsened with time: the higher
tortion due to juvenile fluke hooking was seen in                   the initial infective dose, the more rapid the
the extrahepatic bile duct as early as day 1 p.i.,                  pathologic changes— plateauing occurred at ap-
and acute inflammation was clearly observed on                      proximately day 60 p.i. in all infected groups.
day 3 in group 3. Mononuclear cell infiltration                     From day 60 onwards, active inflammation re-
with subacute inflammation was prominent in the                     gressed and mononuclear cells predominated.
extrahepatic bile duct walls of all infected groups                 Plasma cells were observed in the lamina propria
from day 7 and plasma cells were clearly observed                   of the gall bladder and around the extrahepatic
from day 14 p.i. onwards. Epithelial hyperplasia                    bile duct wall. Eosinophils were seen throughout
with atypical epithelial lining and mitotic figures                 the study but there were not as numerous as those
were apparent from day 7 p.i. Epithelial cells                      observed in the liver (Sripa and Kaewkes, 2000b).
whose lining was in contact with the flukes were                    The most obvious histologic changes in the extra-
flattened. Some periductal glandular formation                      hepatic bile duct in chronic opisthorchiasis were
was also seen. Mucus hypersecretion was ob-                         ductal dilatation, periductal fibrosis and some
served as well as an increased number of goblet                     mononuclear cell infiltration with lymphoid ag-
cells. Fibrosis of the bile duct wall, revealed by                  gregation. Fibrosis with mononuclear cell infiltra-
special staining, was clearly seen on day 30 p.i.                   tion was also prominent in the gall bladder wall.
   From day 30 onwards, Opisthorchis eggs were                      By the end of the experiment, histologic changes
found in the faeces of infected hamsters. Mature                    between groups 2 and 3 were similar. Overall,
flukes were in closer contact with the extrahepatic                 these histologic changes were more severe in the
bile duct mucosa than that of the gall bladders                     extrahepatic bile ducts than in the gall bladders,
because of the narrowness of the duct. Notwith-                     for the same dose and duration of infection. The

Fig. 1. Gross appearances of the liver, gallbladder and extrahepatic bile duct in normal uninfected (a) and O. 6i6errini infected
hamsters (group 3) for 30 (b), 90 (c) and 180 days (d). The thickened and dilated extrahepatic bile ducts are prominent features in
infected hamsters (arrow indicates extrahepatic biliary tree).
B. Sripa, S. Kaewkes / Acta Tropica 83 (2002) 29–36                                       33

Fig. 2. Photomicrograph of normal (a) and O. 6i6errini-infected gallbladder for 7 (b), 30 (c) and 180 days (d). Mononuclear cell
infiltration is gradually observed in the lamina propria, muscle and subserosa. The gallbladder wall is thickened and shows fibrosis
(H&E, original magnification, ×100).

number of mast cells, as revealed by alcian blue–                    trahepatic bile duct caused by O. 6i6errini
PAS-staining, increased in both the gall bladder                     infection.
and the extrahepatic bile duct wall, especially in                      Our study has shown that the morphologic
areas where mucus secretion was prominent. No                        changes in the gall bladder and extrahepatic bile
egg or fluke granuloma were observed in the gall                     duct occurred as early as day 1 p.i. when the
bladder or extrahepatic bile duct in any of the                      juvenile fluke’s sucker caused epithelial distortion.
hamsters. Representative photomicrographs of                         During the early phase of infection, the histologi-
the histologic changes in the gall bladder and                       cal changes, including acute or subacute inflam-
extrahepatic bile duct are shown in Fig. 2a– d and                   mation, edematous villi, increased mucus
Fig. 3a–d, respectively.                                             secretion and goblet cell metaplasia, were dose-de-
                                                                     pendent because hamsters in groups 2 and 3 de-
                                                                     veloped more severe lesions than those in group 1.
4. Discussion                                                        The acute phase (up to day 30 p.i.) might be the
                                                                     result of mechanical irritation and/or a toxic se-
   Opisthorchiasis is a major public health prob-                    cretion from the liver flukes (Bhamarapravati et
lem in Southeast Asia, especially in Northeast                       al., 1978; Hou, 1955). This hypothesis is sup-
Thailand. It is associated with several hepatobil-                   ported by the more severe lesions in the extrahep-
iary diseases. The pathologic consequences of the                    atic bile duct caused by the greater numbers of
infection in the liver are well recognized. How-                     flukes in close contact with the mucosa than was
ever, the histological changes in the gall bladder                   observed in the gall bladder. The toxins released
in opisthorchiasis are only briefly mentioned as                     by O. 6i6errini have not yet been identified.
‘severe inflammation’ in the literature (Riganti et                     Besides the mechanical and/or toxic substances
al., 1989). The present study has described the                      released from the parasite, the immunological
morphologic changes of the gall bladder and ex-                      mechanisms associated with liver fluke infection
34                                     B. Sripa, S. Kaewkes / Acta Tropica 83 (2002) 29–36

may also provoke the reaction (Bhamarapravati et                      onward), the inflammation of the gall bladder and
al., 1978; Flavell and Flavell, 1986; Sripa and                       extrahepatic bile duct was more pronounced. It
Kaewkes, 2000b). Sripa and Kaewkes (2000b) re-                        reached a plateau at the 2nd month p.i. and then
cently demonstrated Opisthorchis antigens in in-                      regressed slightly in all three infected groups. The
fected biliary epithelial cells was associated with                   well-established chronic extrahepatic lesions, in-
inflammation as early as day 3 p.i. Severe inflam-                    cluding prominent fibrosis and some mononuclear
matory cell infiltration, especially with lymphocytes                 cell infiltration, suggests that immunomodulation
and young plasma cells around the biliary tree from                   may occur, particularly in groups 2 and 3. This is
day 14 p.i. onwards in this study, suggests that a                    irrespective of the intensity of the infection. Im-
specific immune response to the liver fluke infection                 munosuppression, both the humoral and cellular
may take place. Reduced inflammatory cell re-                         responses, has been observed in chronic moderate
sponse has been observed in T-cell-deprived ham-                      and heavy O. 6i6errini-infected hamsters (Sirisinha
sters infected with O. 6i6errini compared with                        et al., 1982; Wongratanacheewin et al., 1987).
non-infected hamsters (Flavell and Flavell, 1986).                       Fibrosis of the gall bladder and extrahepatic
Infiltrated mononuclear cells can produce several                     bile duct was similar to that previously described
toxic oxygen-free radicals, such as nitric oxide (NO)                 in the intrahepatic bile ducts in opisthorch-
which may orchestrate the pathologic reactions                        iasis. Periductal fibrosis of the intrahepatic bile
(Miller and Britigan, 1997). Increased NO-syn-                        ducts is a common feature of chronic Opisthorchis
thase, which can catalyze NO, have been found in                      infection, both in animals and man. It has been
eosinophils, macrophages and mast cells of O.                         demonstrated by histological (Bhamarapravati et
6i6errini-infected hamsters (Ohshima et al., 1994).                   al., 1978; Riganti et al., 1989), biochemical
   In chronic O. 6i6errini infection (from day 30                     (Hutadilok and Ruenwongsa, 1983) or radiologi-

Fig. 3. Histology of the extrahepatic bile duct of hamster infected with 50 metacercariae for 3 (a), 14 (b), 30 (c) and 180 days (d).
Active inflammation is seen as early as day 3 p.i. and rapidly increase through day 30 p.i. (c) shows globet metaplasia with mucus
hypersecretion. Dilatation of the lumen, periductal fibrosis and mononuclear cell infiltration with lymphoid aggregation are
prominent features in chronic infection (d). Note mature flukes are also seen in contact with the atrophic mucosa in the lumen (d).
[H&E, original magnification, (a –c) ×100, (d) ×40)].
B. Sripa, S. Kaewkes / Acta Tropica 83 (2002) 29–36                                        35

cal studies (described as enhanced portal vein               for their help in editing the final draft and the
radicle echoes) (Haswell-Elkins et al., 1991; Mairi-         revised manuscript, respectively.
ang et al., 1992). However, no report so far
describes      histopathological       changes     in
opisthorchiasis other than inflammation of the               References
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