The in vitro effects of serum on the adults, metacercariae, and eggs of Opisthorchis viverrini

825 TRANSACTIONS OFTHEROYALSOCIETYOFTROPICAL MEDICINE AND HYGIENE, VOL. 75, No. 6, 1981

The in vitro effects of serum on the adults, metacercariae, Opisthorchis viverrini

and eggs of

D. J. FLAVELL Dept. of Medical

Helminthology, London School of Hygiene and Tropical Medicine, Station, 395 Hatfield Rd., St. Albans, Herts AL4 OXQ, UK

Summary

Adult and juvenile Opisthorchis viverrini liverflukes incubated in hamster chronic infection serum (CIS) developed tegumental precipitates which enclosed the entire parasite. Adult worms appeared dead by 48 hours in CIS and juvenile worms by 18 hours. No such reactions were observed in normal hamster serum (NHS). Heat inactivation of CIS at 56°C abolished the precipitin reactivity against both adult and juvenile parasites though reactivity was regained upon addition of NHS or guinea-pig serum. Reactivity was also abolished by heat inactivating CIS at 60°C but reactivity was not regained upon addition of NHS or guinea-pig serum. Coarse granular precipitates and blebs were observed around eggs in CIS and this activity was abolished by heat inactivating serum at 60°C but not 56°C. Aging of adult parasites in vitro for nine or 27 days before testing against CIS did not affect the development of tegumental precipitates although freeze-thaw killing of adult parasites before testing against CIS resulted in a marked reduction in the extent of the tegumental precipitate reaction. These results show that serum from 0. viverriniinfected hamsters contains a component(s), possibly a specific immunoglobulin(s) capable of reacting with adult, juvenile and egg state parasitic antigens. These findings are discussed in relation to immunopathological processes that might be operating in the liver of the infected host. Introduction viverrini is a liver-fluke

of man found on an endemic scale in the population of north-east Thailand (HARINASUTA & VAJRASTHIRA, 1957). Infections with even large numbers of worms which inhabit the intra- and extra-hepatic bile-ducts, rarely result in acute clinical manifestations, although chronic persistent infections with large numbers of worms can lead to chronic relansing cholangitis and obstructive jaundice (HARI$ASUTA, 1978) and may in some cases eventually lead to cholangiocarcinoma (SONAKUL et al., 1978). In human opisthorchiasis, inflammatory infiltration of bile-duct walls by mononuclear cells and eosinophils is a common finding (KOOMPIROCHANA et al., 1978). Similar reactions have also been observed in experimentally infected hamster hosts Opisthorchis

(BHAMARAPRAVATI

et al.,

1978;

FLAVELL

et al.,

1980b). In hamsters granulomatous-type responses to parasitic eggs gaining accessto portal tract tissues

Winches Farm Field

through breaks in the bile-duct wall have been noted (FLAVELL et al., 1980b). These types of lesion suggest that immunopathological processes may possibly be operating in this helminthiasis. This paper describes the effects of serum taken from hamsters chronically infected with 0. viverrini on the metacercarial, adult and egg stages of the parasite maintained in vitro and shows that such serum contains precipitins against adult, juvenile and egg stage parasites capable of killing parasites in vitro. Experimental

Materials infections

and Methods

A group of 40 male golden Syrian hamsters each initially received 50 0. viverrini metacercariae per OS as described previously (FLAVELL et al., 1980b) and thereafter a further five metacercariae at 14-day intervals until killed at 36 weeks. Each animal therefore received a total of 140 metacercariae over a 36-week veriod. Animals were housed five to a cage on wood shavings and fed a standard hamster chow diet and water ad libitum. Sera Chronic infection serum (CIS) was obtained from the above group of animals at death. Blood was collected into individual alass tubes bv decavitation under deep ether anaesthcksiaand allowed to clot at room temperature. Serum was drawn off when the clot had retracted, serum samples were pooled and stored in sealed containers at - 20°C. until required. Normal hamster serum (NHS) was similarly obtained from a group of 20 overtly healthy animals. Adult and juvenile 0. viverrini parasites Adult 0. viverrini parasites were obtained from

the bile-ducts of hamsters infected six months previously with 50 metacercariae as described previously (FLAVELL et al. 1980a). Only undamaged worms were selected for use after careful microscopic examination. Worms were washed three times in serum-free Earle’s lactalbumin hvdrolvsate (ELAC) medium (Flow Laboratories, Irvine, Scotland) containing 20 mM HEPES and 100 I.U. penicillin and 700 pg streptomycin/ml. For experiments employing parasites aged in vitro, 50 freshly obtained mwormswere transferred to sterile culture flasks containing 10 ml culture medium. Culture fluids were changed once every 48 hours and numbers of parasites removed after nine and 27 days for testing against CIS. Fresh worms for immediate use were held in ELAC at room temperature for no more than two hours before use.

In vitro

826

EFFECTS

OF SERUM

Table I-Reactions of adult worms, juvenile juvenile worms incubated in CIS and NHS

Incubated 10% 10% 50% 10% 10% 5oyo 50% 50%

NHS 56°C NHS CIS 56°C CIS 56°C 60°C

in HI NHS HI CIS HI CIS HI CIS

Adult parasite PPt + +++ -

worms

Juvenile parasite PPt

ND ND +++ -

ON

0. viverrini

and eggs with

Egg PPt trace + +:+ ++

survival

times

Medium PPt + +++ -

of adult

and

Survival* time (days) 10 (2) 10 (2) 10 (2) 10 10 2 (1) 10 (2) 10 (2)

CIS: Chronic infection serum; NHS: Normal hamster serum; HI: Heat inactivated *Figure in parentheses refers to survival time of juvenile worms. Table II-Reactions of adult worms, juvenile worms and eggs with survival times of adult and juvenile worms incubated in CIS heat inactivated at 56°C and 60°C reconstituted with NHS and GE’S

Incubated 50% + 50% + 50% + 50% +

in

56°C HI CIS 25% NHS 56°C HI CIS 25% 56°C HI NHS 60°C HI CIS 25% NHS 56°C HI CIS 25% GPS

Adult Parasite PPt +++

Juvenile Parasite PPt +++

-

Survival* time (days)

Egg PPt

Medium PPt

++

++

2 (1)

-

-

10 (2)

-

-

-

-

10 (2)

+++

+++

+++

++

2. (1)

CIS : Chronic infection serum; NHS : Normal hamster serum; GPS : Guinea pig serum : HI : Heat inactivated *Figure in parentheses refers to survival time of juvenile worms.

Metacercariae were obtained by enzymatic digestion of infected cyprinoid fish in 1.5% pepsin solution (pH 2.5). Metacercariae were recovered from the digests using graded sieves and washed three times in isotonic saline. Freshly excysted juvenile worms were obtained by gently pressing cysts between glass coverslips in culture medium. Juvenile worms obtained in this way were washed three times in serum-free ELAC and stored in medium at room temperature. Experimental protocol Adult worms, newly excysted juvenile worms and metacercariae were tested against two concentrations of untreated and heat-inactivated (56°C and 60°C) CIS and NHS as shown in Tables I and II. Sera were diluted with the appropriate amount of ELAC to give the desired concentration. Additionally, experiments were conducted where heat-inactivated CIS and NHS were reconstituted with 25% NHS or guinea-pig serum as shown in Table II. Circum-oral precipitate (COP) assay Single worms were placed in a migration plate well (Sterilin Ltd., Richmond, Surrey, UK) and

the appropriate culture fluid added. Wells were sealed with sterile coverslips (22 x 22 mm) held in place with silicone grease (Edwards High Vacuum, Crawley, Surrey, UK) and incubated at 37°C. Parasites in individual wells were observed under low power of an inverted microscope at 2, 6, 18 and 24 hours and then once daily for a further nine days. An arbitrary scoring system providing a semiquantitative assessment of the extent of precipitates appearing at the parasite surface, around eggs or in the culture fluids was constructed. Thus, maximal precipitate formation as shown in Fig. 1 was scored as + + + and minimal as shown in Fig. 6 as +. Parasites which lost all apparent locomotory movements, gut movements and their characteristic translucent appearance were judged dead. Parasites were scored as moribund when they experienced obvious difficulty in moving or when such movements were feeble and/or erratic. Results Adult and juvenile worm reactions The results for reactions of adult and juvenile worms incubated in the various sera are sum-

D. J. FLAVELL

827

Fig. 1. A moribund adult 0. viverrini parasite after 18 hours’ incubation in 50% CIS showing membranous-type precipitate enclosing the anterior portion of the worm. x 25

Fig. 2. Filamentous-type precipitate “growing out” from the tegumental surface of an adult parasite incubated for 18 haul rs in 50% CIS. x 185

Fig. 3. Precipitous CIS. x 185

Fig. 4. 0. viverrini dense precipitate

mass

associated with

parasitic

eggs in 50%

eggs incubated in 50% CIS showing surrounding with large bleb-like protuberance. X 350

In

vitro

EFFECTS

OF SERUM

Fig, 5. Large bleb-like masses shedding from the tegument surface of an adult parasite incubated for 48 hours in 50% CIS. x 185

Fig. 7. Metacercariae reactivity. x 80

incubated

in 50%

CIS

showing

lack of

ON

0.

ViVeWini

Fig. 6. A freeze-thaw killed adult parasite incubated in 50% CIS showing small tufts of precipitate at the tegument surface. x 185

Fig. 8. NewIy excysted juvenile worm incubated in 50% CIS for 4 hours showing early precipitate formation. x 185

829

D. J. FLAVELL

marized in Table I. A total of 16 adult worms, 20 juvenile worms and 50 metacercariae were tested against each serum preparation. All 16 adult parasites incubated in 50% CIS developed + + + tegumental precipitates by 18 hours in culture and usually precipitates were demonstrable after four to six hours in culture. The precipitate masses invariably enclosed the entire parasite as sheath-like structures and generally were evenly distributed over the entire parasite surface. These tegumental precipitates took two forms, either (i) a translucent membranous type precipitate (Fig. 1) or (ii) an opaque filamentous type precipitate which appeared to “grow out” from the tegumental surface (Fig. 2). All parasites appeared moribund after 18 hours in 50% CIS and the nreciuitates auoeared to restrict-parasite movement. gy 48 hours all 16 parasites in 50% CIS appeared dead. Ten of the flukes incubated in 50% CIS developed large surface blebs over some areas of the tegument (Fig. 5). In 10% CIS tegumental precipitates scored as + appeared after 18 hours but the extent and intensity of the reaction was markedly below that encountered in 50% CIS. Moreover, all narasites were alive and apparently healthy after lo-days in 10% CIS. No’tegumental precipitates were observed around flukes incubated in NHS and all parasites survived for more than ten days. Heat inactivation of CIS at 56°C for 30 min abolished the precipitin and parasiticidal activities of the serum (Table II). However, both activities were regained upon addition of 25% NHS or guinea-pig serum (Table II). Heat inactivation of CIS at 60°C similarly abolished reactivity of serum against adult worms in culture, but reactivity was not regained upon addition of NHS or guinea-pig serum. No reaction was observed when intact living metacercariae were incubated in 50% CIS (Fig. 7) and all metacercariae were alive ‘after 10 days incubation. When newly excysted juvenile flukes were incubated in 50% CIS tegumental precipitates appeared within two to four hours (Fin. 8) and all worms appeared dead by 18 hours. .This reactivity was abolished by heat inactivation at 56°C and was regained upon the addition of NHS or guinea-pig serum. No reactions were seen when juvenile flukes were incubated in NHS though most of the juvenile worms survived no longer than two days in NHS. Egg reactions Reactions of eggs secreted by adult parasites in vitro in the various sera are summarized in Tables

Table III-Reactions of adult incubation in 50% CIS Adult Parasite source

Aged 9 days in culture Aged 27 days in culture Freeze-thaw killed parasites

worms

I and II. In both 10% and 50% CIS, dense, semitranslucent precipitates were seen to surround the entire egg and bleb-like protuberances were frequently seen to extend outward from the main precipitate mass (Fig. 4). Heat inactivation of CIS at 60°C but not 56°C abolished the precipitin reactivity against eggs (Table I). Large granular precipitous masses were frequently seen distributed throughout wells containing adult flukes in 50% CIS and these precipitates were frequently, though not invariably associated with parasite eggs (Fig. 3). However, heat inactivation of CIS at 56°C resulted in the total loss of the large granular precipitous masses appearing! in the medium without loss of egg precipitin reactivity as shown in Fig. 3. In NHS very weakly defined precipitates were observed around a few eggs. These were transparent and no more than 1 to 2 p thick. Reactions of dead and aged parasites Results obtained when dead parasites or those aged in vitro were tested against CIS are shown in Table III. The maintenance of adult worms in vitro for nine and 27 days before testing against CIS did not affect the extent or intensity of the observed tegumental precipitates developing after incubation in CIS (Table III). Similarly, egg precipitates and large granular precipitous masses seen in cultures of aged worms incubated in CIS were identical to those observed when fresh parasites were employed in the test system. When freeze-thaw killed parasites were incubated in 50% CIS for four to six hours, a slight precipitous reaction occurred at the tegument surface with small amounts of dense material appearing as tufts at the parasite surface (Fig. 6). However, this reaction did not proceed any further and precipitous material did not appear in the culture fluids. Discussion The present study clearly shows that serum from 0. viverrini chronically infected hamsters (CIS) contains a component(s) giving precipitous reactions with juvenile, adult and egg stages of the parasite. Moreover, CIS killed adult parasites within 48 hours and juvenile parasites within 24 hours. Intact metacercariae did not react with CIS. The nature of the serum factor(s) responsible for the precipitin and/or parasiticidal effects has not been established in this study though, on the basis of the heat inactivation studies, it seems probable that it is a specific immunoglobulin(s). Both precipitin and parasiticidal activities of CIS

and eggs aged in vitro or killed

by freeze-thawing

Parasite PPt

Egg PPt

Medium PPt

Survival time (days)

+++ +++ No eggs

++ ++ -

2 2 -

+++ +++ +

before

830

In Vitro EFFECTSOF SERUM ON 0. viverrini

against adult and juvenile worms appear to be complement dependent, activity being completely abolished by heat inactivation of CIS at 56°C but regained upon addition of NHS or guinea-pig serum as a source of complement. This observation is in sham contrast to the studies of SUN (1969a) who showed that the precipitin and parasiticida activities of serum against the closely related liverfluke Clonorchis sinensis were unaffected by heat inactivation of serum at 56°C. On the basis of the heat inactivation studies, the parasite egg precipitates observed with CIS would appear to be complement independent. The bleblike protuberances seen to extend outwards from the main egg precipitate mass suggest that the putative ant;genic component(s) is asecretory egg vroduct. SUN (1969b) and SUN & GIBSON(1969a) were “unable ‘to demonstrate serum precipitin reactions against C. sine&s eggs and the results of the present study would suggest a major difference between 0. viverrini and C. sinensis with regard to egg antigenicity. Granulomatous reactions to 0. viverrini eggsin the livers of experimentally infected hamsters have been noted previously (FLAVELL et al., 1980b; BHAMARAPRAVATI et al., 1978) and such host responses may represent specific host immunological reactions to eggs. SUN 11969a) considered that metaboliclsecretorv products of adult C. sinensis worms formed the putative antigenic component(s) reacting with immune serum. SUN & GIBSON (1969a, b) have demonstrated that sera from several different C. &ens&infected mammalian hosts contain precipitins against parasitic metabolic/secretory products using immunodiffusion and that the antigenic activity of such secretions appears to be associated with digestive enzymes of the parasite (SUN & GIBSON, 1969b). The observation of the present study that the freeze-thaw killing of adult parasites results in a marked reduction in the extent and amount of tegumental precipitate appearing in CIS implies that intact parasitic metabolic processesare required for the reaction to proceed to a maximal extent. Maintenance of adult parasites for up to 27 days in vitro before testing against CIS did not, however, result in impairment of tegumental precipitate appearance. The appearance of large bleb-like bodies at the tegumental surface of adult parasites incubated in CIS may represent an attempt by the parasite to shed portions of the tegument affected by immune precipitate or alternatively may indicate structural damage at the fluke surface. The killing of parasites in CIS may result directly from antibody reacting with specific antigen(s) at the parasite surface eliciting complement mediated damage to the tegument. Alternatively, the formation of dense precipitates around parasites may impede or prevent normal nutritional, excretory and secretory parasite functions, causing death. Immunologically the bile-duct lumen avoears to be a fairly in&t site and the question as to whether the in vitro varasiticidal effects of CIS described here bears any relevance to the killing of worms in vivo remains unresolved and subject to further investigation. However, putative parasitic antigens

in the form of soluble somatic components, excretory/secretory products of eggs may gain access to portal tract areas through breaches in the bile-duct wall or by simple diffusion and may elicit antibody production or cell-mediated immune reactions. The histovatholonical resoonses of the host to 0. viverrini infection include inflammatory reactions in portal tract regions sometimes extending deep into the parenchymal tissues and granulomatoustype reactions around eggs that gain access to the liver tissues proper (FLAVELL et al., 1980b). The occurrence of such reactions indicates that immunopathological processes may be operating in the liver of the infected host. The preliminary observations described in the present study show that presumptive host antibody is capable of reacting with putative antigen(s) of juvenile, adult and egg stages of the parasite warranting further investigation into the nature of the antigen(s) and their possible role in the pathology of the disease.

Acknowledgements

The author gratefully acknowledges the assistance of Miss S. Tranfield with preparation of photographic materials and also Mr. Kovit Pattanapanyasat and Mrs. Malulee Tuntawiroon, Bangkok, for the provision of infected material. This work was generously supported by a grant from the Wellcome Trust. The author is a Wellcome Research Fellow. References

Bhamaraparavati, N., Thamavit, W. & Vajrasthira, S. (1978). Liver changes in hamsters infected with a liver fluke of man, Opisthorchis viverrini. American Journal of Hygiene, 27, 787-794.

Tropical

Medicine

and

Flavell, D. J., Pattanapanyasat, K. & Flavell, S.U. (1980a). Opisthorchis viverrini: Partial success in transferring immunity with spleen cells and serum in the hamster. Journal of Helminthology, 54, 191-197. Flavell, D. J., Pattanapanyasat, K., Lucas, S. B. & Vongsangnak, V. (1980b). Opisthorchis vzverrini: Liver changes in golden hamsters maintained on high and low protein diets. Acta Tropica. - _ 37., 337-350. Harinasuta, C. (1978). Opisthorchiasis in Thailand. South East Asian Journal of Tropical Medicine and Public Health, 9, 281. Harinasuta. C. & Vairasthira. S. (1957). Studv on opisthorchiasis in ‘Thailand. I: Survey of- the incidence of the infection in patients of fifteen hospitals in the north-east. Proceedings of the 9th Pacific Science Congress, Bangkok, p. 204. Koompirochana, C., Sonakul, D., Chinda, K. & Stitnimankarn, T. (1978). Opisthorchiasis: a clinicopathologic study of 154 autopsy cases. South East Asian Journal of Tropical Medicine and Public Health. 9, 60-64.

Sonakul, D., Koompirochana, C., Chinda, K. & Stitnimankarn, T. (1978). Hepatic carcinoma with opisthorchiasis. South East Asian Journal of Tropical Medicine and Public Health, 9, 215219.

831

D. J. FLAVELL

Sun, T, (1969a). The in vitro action of antisera on the adults of Clonorchis sinensis. Transactions of the Royal Society of Hygiene, 63, 582-590.

Tropical

Medicine

and

Sun, T. (1969b). The non-antigenicity of intact ova of Clonorchis sinensis. Japanese Journal of Medical Microbiology,

American Journal of Hygiene, 18, 241-252.

Tropical

Medicine

and

Sun, T. & Gibson, J. B. (1969b). Metabolic products of adult Clonorchis sinensis: Their composition and antigenic potential. Journal of Helminthology, 43, 395-402.

2, 358-360.

Sun, T. & Gibson, J. B. (1969a). Antigens of Clonorchis sinensis in experimental and human infections. An analysis by gel diffusion technique.

Accepted for publication 14th March, 1981.

I

1 INTERNATIONAL

SOCIETY

AND FEDERATION 1982

OF CARDIOLOGY

1982 has been declared the Year of Tropical Cardiology. It is hoped that Schools of Hygiene and Tropical Medicine, as well as Hospital Centres, throughout the world will organize symposia and/or workshops in commemoration. Although the title may suggest that only tropical cardiac diseasesare considered relevant, other heart diseases (e.g., rheumatic fever, hypertension, ischaemic heart disease and cardiomyopathies) of worldwide distribution, as seen in the tropics or subtropical regions, are included. For information please contact E. G. J. Olsen, MD, FRCPath, FACC, The National Heart Hospital, Westmoreland Street, London WIM 8BA or the Central Office of the International Society and Federation of Cardiology, 34, rue de l’Athenee, P.O. Box 117, CH-1211, ,Geneva 12, Switzerland.