Gall bladder and extrahepatic bile duct changes in
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Acta Tropica 83 (2002) 29 – 36
www.parasitology-online.com
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.
PII: S 0 0 0 1 - 7 0 6 X ( 0 2 ) 0 0 0 5 2 - 930 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 infection34 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
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radicle echoes) (Haswell-Elkins et al., 1991; Mairi- revised manuscript, respectively.
ang et al., 1992). However, no report so far
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