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Keywords:

  • Opisthorchis viverrini;
  • antibody responses;
  • ELISA;
  • worm burden;
  • EPG;
  • hamsters

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The kinetics of parasite-specific antibody responses in relation to worm burden and egg output were investigated in hamsters infected with 25, 50 and 100 Opisthorchis viverrini metacercariae (MC). Levels of antibody to egg, excretory–secretory (ES) and somatic antigens were examined by ELISA on days 1, 3, 7, 14 and month 1 postinfection (p.i.), and repeated monthly up to 6 months. The antibody responses were first detected as early as 14 days after infection. Hamsters that were infected with 100 MC and 50 MC showed higher antibody levels than those of 25 MC, during early infection until 1 month p.i. Then, the antibody levels were increased rapidly to a plateau at approximately month 2 p.i. and, subsequently, were relatively stable in all groups. The average antibody levels to egg and somatic, but not to ES antigens, were significantly higher in hamsters infected with 25 MC than those of 50 MC and 100 MC. These antibody responses, particularly to egg and ES antigens, were not correlated with worm burden or egg output. Overall, higher antibody responses were found in the order: ES, somatic and egg antigens. The significant lower antibody responses in chronic and heavy infections than those with mild infection may a result of immunosuppression.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The liver fluke infection caused by Opisthorchis viverrini, O. felineus and Clonorchis sinensis remains a major public health problem in many parts of Eastern Europe, the Far East and South-east Asia ( IARC 1994). In North-east Thailand, which is an endemic area of opisthorchiasis, an estimated 3.8 million people (calculated from 18.5% prevalence in this region) are infected with O. viverrini ( Jongsuksuntikul & Imsomboon 1997). This infection is associated with a number of hepatobiliary diseases, including cholangitis, obstructive jaundice, hepatomegaly, cholecystitis and cholelithiasis ( Harinasuta et al. 1984 ). Moreover, both experimental and epidemiological evidence strongly implicate the liver fluke infection in the aetiology of cholangiocarcinoma, the bile duct cancer ( Thamavit et al. 1978 , Haswell-Elkins et al. 1992 , Sithithaworn et al. 1994 , Elkins et al. 1996 ).

The pathological consequences of O. viverrini infection appear to be similar in both human and animals. These include epithelial desquamation, inflammation, epithelial hyperplasia, goblet cell metaplasia, adenomatous hyperplasia and periductal fibrosis ( Bhamarapravati et al. 1978 , Riganti et al. 1989 ). Several investigators have suggested that immunopathological processes may play a major role in the liver fluke-associated liver damages ( Bhamarapravati et al. 1978 , Flavell & Flavell 1986, Haswell-Elkins et al. 1991 ). Significant association between periductal fibrosis, as revealed by enhanced portal vein radicle echoes in heavy infected people and high O. viverrini-specific IgG levels, has been described ( Haswell-Elkins et al. 1991 ).

To elucidate immunopathological processes, immune responses to O. viverrini infection should be fully characterized. Several papers on enhanced immune responses to infection both in human and animal models have been published previously ( Janechaiwat et al. 1980 , Sirisinha 1986). These responses declined after elimination of the liver flukes by praziquantel treatment ( Wongratanacheewin & Sirisinha 1987, Thammapalerd et al. 1988 , Wongratanacheewin et al. 1988a , Ruangkunaporn et al. 1994 ). Only two papers have described the kinetics of antibody responses, which may reflect immunopathological processes more precisely than a study at a time-point ( Sirisinha et al. 1983 , Chawengkirttikul & Sirisinha 1988). Sirisinha et al. (1983 ) showed decreased antibody levels to both somatic and excretory–secretory (ES) antigens in chronic and heavy infections. Similar results were also observed for somatic antigens ( Chawengkirttikul & Sirisinha 1988). Surprisingly, there are no reports describing levels of antibody response to egg antigens, although antibody responses to parasite eggs have been demonstrated by immunofluorescence and immunoradiography ( Wongratanacheewin & Sirisinha 1987) and study in vitro ( Flavell 1981). The objective of the present study was to investigate the kinetics of humoral immune responses to egg, ES and somatic antigens in relation to the intensity of O. viverrini infection.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Parasite

Metacercariae were obtained from naturally infected cyprinoid fish from an endemic area in Khon Kaen Province, Thailand by pepsin–HCl digestion and filtration. The larvae were washed several times with normal saline and collected under a dissecting microscope.

Preparation of parasite antigens

Adult O. viverrini worms were obtained from the livers and bile ducts of hamsters infected for 3–4 months previously. The fresh worms were washed several times in cold normal saline containing penicillin (200 U/ml) and streptomycin (200 U/ml) to remove any debris and residual blood. After washing thoroughly, the viable worms were used for the collection of ES products and the inactive ones were quick frozen in liquid nitrogen and stored at −80°C prior to homogenization. For somatic antigens, the frozen worms were crushed and ground in liquid nitrogen. The ground powder was then dissolved in PBS containing protease inhibitors (0.1 m m PMSF, 1 m m EDTA, 1 m m leupeptin and 0.1 m mN-[N-( l-3-trans-carboxyoxiran-2-carbonyl)- l-leucyl]-agmatine, E-64) ( Itoh et al. 1990 ). The suspension was then sonicated, centrifuged at 10000 r.p.m. for 30 min at 4°C and the supernatant stored at −80°C.

ES antigens were prepared by in-vitro culture of viable flukes in the modified Tyrode's buffer containing penicillin (100 U/ml) and streptomycin (100 U/ml), 0.1 m m PMSF, 1 m m EDTA, 1 m m leupeptin and 0.1 m m E-64 at 37°C for up to 5 days. Dead worms were periodically removed. The culture fluid was changed every 24 h, pooled and centrifuged to remove the eggs. The supernatant and pellets were stored at −80°C for preparing ES and egg antigens, respectively. For ES antigens, the pooled supernatant was concentrated by membrane ultrafiltration (PM 10, Amicon, Danver, MA, USA), dialysed in PBS several times and then aliquoted at −80°C.

For egg antigens, eggs were washed several times in normal saline to remove any residual ES products. After centrifugation, the egg pellets were crushed in liquid nitrogen as above and aliquoted at −80°C.

Protein concentrations of all O. viverrini antigen preparations were determined by the Bradford method (Bio-Rad Hercules, CA, USA).

Animals and experimental design

Two hundred male Syrian golden hamsters (Animal Unit, Faculty of Medicine, Khon Kaen University), aged 6–8 weeks, were used. The hamsters were divided into four groups of 50; groups 1, 2 and 3 were infected with 25, 50 and 100 metacercariae by intragastric intubation, respectively. Group 4 was given normal saline. The animals were housed under conventional conditions, fed a stock diet and water ad libitum. The maintenance and care of experimental animals complied with the National Experimental Animal Center guidelines. Five animals from each group were sacrificed on days 1, 3, 7, 14 and 30 (month 1) p.i. The rest of the animals were killed monthly up to 6 months.

Surgical procedures and sample collection

The hamsters were weighed, terminally anaesthetized and bled from the heart. Serum was stored at −20°C for antibody assays. The liver was dissected, weighed and kept in normal saline for worm counting.

Determination of egg and worm counts

The animals were kept singly for 3 days before sacrificing to collect faeces. Number of eggs per gram (EPG) were determined by the quantitative formalin/ethyl acetate concentration technique of Elkins et al. (1986 ). Worm burdens were determined by counting worms in liver squashes.

Enzyme-linked immunosorbent assay (ELISA)

O. viverrini-specific IgG was determined by avidin–biotin ELISA. Dilutions of serum, biotinyated antibody and strepavidin–peroxidase conjugate in each assay are shown in Table 1. Wells were coated with 100 μl of ES, somatic or egg O. viverrini antigen (1 μg/ml in 0.05 m carbonate buffer, pH 9.6) overnight at 4°C. The plates were then washed three times with 0.05% Tween 20 (Amresco, OH, USA) in normal saline (NSST). Eeach well was blocked with 200 μl 5% skimmed milk in 0.05 m carbonate buffer (pH 9.6) for 60 min at 37°C. After washing with NSST, the wells were incubated with 100 μl of hamster serum, in duplicate, in 2% skimmed milk in an incubation buffer (PBS with 0.05% Tween) for 60 min at 37°C. After washing, 100 μl of biotinylated antihamster IgG (Vector Laboratories. Inc., Burlingame, CA, USA) in 2% skimmed milk in incubation buffer and strepavidin–peroxidase conjugate in PBS were sequentially applied to each well and each was incubated for 60 min at 37°C. After extensive washing, 100 μl of freshly prepared OPD substrate solution (Dako, Glostrup, Denmark) was added and the reaction was stopped with 100 μl 4 n sulphuric acid. Optical densities (OD) were read at 490 nm. The readings were adjusted for daily variation by dividing the OD of each test by the mean value for positive control sera. Between test variability was minimal with an coefficient variability of less than 10% for each antigen. Normal uninfected hamster sera gave a very low background for the somatic and ES antigens (OD490 of 0.07 ± 0.07 and 0.05 ± 0.05, respectively). Slightly more background was found for the egg antigens (OD490 of less than 0.2).

Table 1. O. viverrini antigen concentrations for plate coating and dilutions of serum samples, biotinylated antibody and strepavidin–peroxidase used in ELISA Thumbnail image of

Statistical analysis

Data were analysed using the statistical software, SPSS/PC+ (Chicago, IL, USA). Transformation of some data, where appropriate, was necessary to obtain normality. A value of P < 0.05 was considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Worm recovery and EPG

The kinetics of the worm recoveries and mean worm recovery rates (%) in the infected groups 1, 2 and 3 are shown in Figure 1. At early time points, worm recoveries were low in all groups, especially in the group that was infected with 25 MC (group 1) because the juvenile flukes are very small. From 14 days p.i. onwards, the worm recovery rate became relatively stable with the overall mean worm recovery rates of 50%, 55.2% and 46.7% for groups 1, 2 and 3, respectively. These worm recovery rates were not statistically significant among the three infected groups during the course of the experiments.

image

Figure 1 Mean worm burdens and recovery rate (%) in hamsters infected with 25, 50 and 100 O. viverrini metacercariae from day 1. to month 6 postinfection. D, day; M, month.

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Parasite eggs in the faeces were first seen at month 1 p.i. in all infected hamsters of group 2 and 3 but in two out of five hamsters of group 1. The EPG was low at month 1 p.i., then gradually increased to a peak at month 4 for groups 2 and 3 (mean EPG = 1839 and 2649.6, respectively) and month 5 for group 1 (mean EPG = 2461.2). Significant reduction of the mean EPG was seen after the peaks in groups 2 and 3 but not in group 1. Overall, no correlation between worm burden and corresponding EPG was found (Pearson correlation, r = −0.075, P > 0.05). An inverse correlation was observed between the mean EPG/worm and worm burden (r = −0.542, P < 0.01). The mean EPG/worm of group 1 was significantly higher than those of groups 2 and 3 during the course of infection ( anova, P < 0.05). The EPG/worm was highly correlated with the corresponding EPG (r = 0.744, P < 0.01). The kinetics of the mean EPG and EPG/worm at different time points is shown in Figure 2.

image

Figure 2 Time-course of egg output (EPG) and EPG per worm in hamsters infected with 2. 5, 50 and 100 O. viverrini metacercariae from month 1 to month 6 postinfection. M, month.

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Antibody responses to parasite antigens

The kinetics of serum antibody responses to O. viverrini antigens are shown in Figure 3(a–c). Antibody responses were first detected at day 14 p.i. The general patterns of responses to egg, ES and somatic antigens were similar. Early antibody responses to all antigens tested were observed more frequently in hamsters infected with 50 MC or 100 MC and were dose-dependent. Antibody responses then increased rapidly to a plateau at one month in groups 2 and 3, but rose more gradually and reached a plateau at month 2 p.i. in group 1. Antibody responses to ES and somatic antigens at 1 month p.i. in groups 2 and 3 were significantly higher than those of group 1 ( anova, P < 0.05). The levels of antibody to egg antigens increased most rapidly in the hamsters of group 2 and were significantly higher than the other groups ( anova, P < 0.05).

image

Figure 3. Kinetics of serum antibody responses to (a) ES, (b) somatic and (c) egg antigens in hamsters infected with 25, 50 and 100 O. viverrini metacercariae from day 1 to month 6 postinfection. Data represent the mean OD (490 nm) of five animals. D, day; M, month.

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From month 2 p.i. onwards, parasite-specific IgG levels to all antigens were relatively stable ( anova, P > 0.05). However, the overall magnitude of antibody responses to egg and somatic antigens was significantly higher in group 1 than in groups 2 and 3 ( anova, P < 0.05 and 0.005, respectively). A similar but not significantly different pattern was seen with the ES antigens.

Relationships between antibody levels, worm burden and EPG

Correlations between the levels of parasite-specific IgG to egg, somatic or ES antigens, worm burden and EPG at different time points were evaluated beginning after 1 month. At 1 month p.i., the levels of anti-ES and antisomatic antibodies were correlated with worm burden for the former (r = 0.519, P < 0.05) and EPG for the latter (r = 0.571, P < 0.05). The most striking finding was the steep elevation of parasite-specific IgG to egg antigens in group 2. The egg output of this group (EPG = 191.6) was higher than those of groups 1 and 3 (EPG = 29.6 and 151, respectively). Moreover, a significant correlation between the mean EPG and anti-egg IgG levels was also found in this group (r = 0.578, P < 0.05). After 1 month, the levels of antibody to each antigen were not correlated with either the EPG or worm burden except for that of antisomatic antigens, which were correlated with the corresponding EPG (r = 0.215, P < 0.05).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The present study has shown that humoral immune responses to egg, ES and somatic antigens in hamsters infected with three different intensities of O. viverrini occur as early as day 14 p.i. Early responses were clearly associated with the intensity of the liver fluke infection, as determined by both worm burden and EPG. However, for chronic infection, there were inverse relationships among parasite-specific antibody levels, intensity of infection and egg production, suggesting immunosuppression.

Analysis of the parasitological parameters revealed that worm recoveries in all infected groups were similar and relatively stable during the course of the study. An interesting point was the significant reduction of EPG and EPG/worm in chronic infection, especially in heavy infection (group 2 and 3). Such findings were also observed by other investigators ( Flavell et al. 1983 , Sirisinha et al. 1983 ). However, our study has extended the course of the infection to 6 months and has provided data for egg counts. Similar relationships between O. viverrini egg output and worm burden have been described in human autopsies ( Sithithaworn et al. 1991 ) and in individuals residing in endemic areas ( Elkins et al. 1991 ). The effects may be related to the competition of parasites for the limited nutrients available within the biliary tree, in the case of O. viverrini, or possibly to the host immune responses. In support of an immunological modulation of egg excretion, Flavell et al. (1983 ) demonstrated that the correlation between worm burden or worm density (worm/liver weight) and egg output/worm was identical. If crowding effects alone were responsible for decreased egg output, then the correlation between egg output and worm density (the true measure of crowding) should be considerably better than that between egg output and worm burden. This finding was also observed in the present study (data not shown).

Evidence supporting the role of immunological response in the reduction of egg output and egg output/worm in chronic infection has been described. Hamsters that were repeatedly infected with O. viverrini ( Sirisinha et al. 1983 ) or receiving either serum or spleen cells from infected hamsters ( Flavell et al. 1980 ) showed a reduction of egg output or EPG per worm. However, Flavell & Flavell (1986), in a subsequent study, failed to demonstrate differences in egg output in T-cell deprived hamsters compared with their immunologically intact counterparts. Therefore, the relationship between reduction of egg output and host immune responses is still unclear.

Analysis of antibody responses to different O. viverrini antigens indicated that early responses were intensity dependent. Responses were highest for ES antigens, moderately high for somatic and lowest for egg antigens. Surprisingly, antibody to egg antigens was detected on day 14 p.i. in groups 2 and 3, before eggs are produced, which may be explained by common components between egg and other antigens of O. viverrini ( Wongratanacheewin et al. 1988b ).

Stimulation of immune responses clearly occurs during both juvenile and adult stages, through the release of antigens into the bile or host tissue. Sun & Gibson (1969) were able to detect parasite antigens in the bile of patients and in animals infected with C. sinensis, the close relative of O. viverrini. Opisthorchis antigens were also demonstrated in the bile of infected hamsters by immunoblotting and ELISA (Sripa & Kaewkes, in preparation). Parasite antigens may enter through the damaged biliary epithelium, as previously described in infected hamsters ( Bhamarapravati et al. 1978 ) and/or by bile penetrating through damaged liver cells as suggested in C. sinensis infection ( Sun & Gibson 1969). However, the periportal or periductal areas with intact epithelium also show heavy mononuclear cell infiltration, which suggests that activation might take place in situ. The inflammatory responses evoked by O. viverrini are immune-mediated, and T-cell deprived hamsters show less inflammation than T cell-sufficient animals ( Flavell & Flavell 1986). Since opisthorchiasis is a mucosal infection, it is unlikely that lymphoid cells come into direct contact with the flukes, but metabolic products or other antigens may diffuse through the biliary epithelium into surrounding tissue. Certainly, ES and somatic antigens have been detected immunohistochemically in the biliary epithelium of the intrahepatic bile duct, gallbladder, extrahepatic bile duct and surrounding tissue, as early as day 3 p.i. in infected hamsters (Sripa & Kaewkes, submitted).

During the chronic phase of infection, antibody responses, after reaching their plateaus, were relatively stable and similar for all antigens tested in all infected groups. Significantly higher responses, particularly to somatic and egg antigens, were found in the lightly infected hamsters compared with those with heavy infection. Continuous release of antigens may result in the eventual suppression of both humoral and cell-mediated immune responses (CMIR). Wongratanacheewin et al. (1987 ) showed that depression of antibody response to sheep red cells and decreased lymphocyte proliferation to PHA occurred in chronically and heavily infected hamsters. In addition, Opisthorchis-infected hamsters have been shown to lose CMIR to the parasite antigens ( Sirisinha et al. 1982 ). In both studies, the depression was abolished and its activity restored after elimination of the liver flukes by praziquantel treatment. The mechanism(s) by which the liver fluke induces immunosuppression is not yet known, but may involve immunoregulation by T-cell subpopulations, as suggested by Wongratanacheewin et al. (1987 ).

In conclusion, the present study has shown that humoral immune responses to egg, ES and somatic O. viverrini antigens were dose-dependent at an early stage of the infection. Significant reductions of egg output and egg output per worm, but not worm counts, in chronic and heavy infected hamsters were inversely correlated with parasite-specific antibodies. Suppressed antibody responses to somatic and egg antigens, but not to ES antigens, were observed during chronic infection, suggesting that immunosuppression did occur. The antibody responses may directly reflect the host pathology. Therefore, hepatobiliary changes should be studied in relation to the immunological status of infected hosts. Since opisthorchiasis is a chronic asymptomatic disease, even in patients with heavy infection ( Upatham et al. 1984 ), the mechanism of immunosuppression is of interest. Particularly, detailed studies on specific T cell subpoulations, i.e. Th1, Th2 and their cytokine production, should be performed to elucidate their roles in immunosuppression and immunomodulation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

This work was supported by the Thailand Research Fund (TRF), grant No. BRG3980013. The assistance of Mrs Aoumporn Mongkolwongroj, Mr Suvit Balthaisong and Miss Wanna Sobai are appreciated. Miss Evelyn L. Murphy is also acknowledged for revising the English grammar.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References
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