Formation of Echinococcus granulosus laminated membrane in a defined medium

lnrernotional fourna~/or Parasitolony. 1976.Vol.6.p~.467-471. PergamonPress. Prinrrd inGreatBrifaln.

FORMATION OF ECHINOCOCCUS GRANULOSUS LAMINATED MEMBRANE IN A DEFINED MEDIUM D. D. HEATH and P. J. OSBORN New Zealand Ministry of Agriculture and Fisheries, Wallaceville Animal Research Centre, Private Bag, Upper Hutt, New Zealand (Received 24 December

1975)

Abstract-HEATH D. D. and OSBORNP. J. 1976. Formation of Echinococcus grunulosus laminated membrane in a defined medium. International Journal for Parasitology 6: 467-471. Eehinoccocus granulosus protoscoleces were digested from brood capsule material using artificial gastric fluid, and were cultured for 60 days in medium NCTC 135. Vesiculation occurred within 7 days, and the first laminated membranes were observed after 17 days of culture. Some contaminating sheep antigens appeared to be lost after 21 days. INDEX KEY WORDS: Echinococcus grarudosus; vitro culture; laminated membrane; host antigens.

INTRODUCTION Echinococcus

granulosus

protoscoleces cultured in various media, all of which contained undefined macromolecules, developed into small cysts with laminated membranes @myth, 1962). He concluded that the laminated membrane was formed initially by the parasite, but there remained some doubt as to whether host-derived macromolecules were required. Varela-Diaz & Coltorti (1973) demonstrated specific 1gG in laminated membranes, and pointed out the relevance of this observation to the hypothesis of Kilejian, Sauer & Schwabe (1962) and Kilejian & Schwabe (1971) that a host factor may be partially responsible for the formation of laminated membranes. The present observation demonstrates the parasite origin and formation of the laminated membrane, and describes a technique whereby cystic E. granulosus material may possibly be obtained with little contamination by host protein. This may be of value for immunodiagnosis of hydatid disease in humans, because false positive reactions have been associated with the presence of sheep serum components in hydatid cyst fluid (Kagan, 1968) or cyst membranes used for antigen production (VarelaDiaz & Coltorti, 1973). MATERIALS

AND METHODS

Brood capsules were obtained using sterile techniques @myth, 1967) from E. granulosus cysts in the livers and lungs of mature Romney sheep. After washing twice in the culture medium (NCTC 135; GIBCO, NY). brood capsule wall was removed by digestion at 37°C for 10 or 60 min with artificial gastric fluid (1 “/, of 1 : 2500 Peosin [B.D.H.] plus 1% of-10 N-HCl in b.857; NaCI). ?he enzyme solution was removed by washing 3 times with NCTC 135 at 37°C. The culture medium was filtered

protoscolex;

vesiculation;

defined medium;

in

under nitrogen through a 0.2 Hrn membrane. Four cultures were established from each incubation time in 250 ml flasks (Falcon Plastics), completely filled with medium which was changed at 3-day and 4-day intervals for 8 weeks. Penicillin G (Sigma) 100 i.u./ml, and streptomycin sulphate (Glaxo) 100 pg/ml, were added for the first 7 days, but omitted thereafter. To determine whether contaminating sheep macromolecules bad been removed by in vitro culture, equal numbers of protoscoleces (2 ml sedimented volume) were treated with artificial gastric fluid for 10 min and then washed as described above. One sample was frozen immediately and stored at -20°C while the other was cultured for 21 days. Each sample was frozen with liquid Nz in a mortar and ground to a slurry, which was further homogenized using a PotterElvehjem glass homogenizer for 2 min at 4°C. The homogenates were then centrifuged at 140000 g for 2 h at 4°C and the supernatants were removed using a Pasteur pipette, taking care to leave the surface lipid layer behind. Any contaminating lipid was then removed by filtration through a 0.2 Mm membrane. The supernatants were then concentrated fourfold in an Amicon Ultrafiltration Cell using a PM10 membrane and stored at 4°C until required. Immunodiffusion and immunoelectrophoresis were conducted using L.K.B. Multiphor apparatus following methods described by Williams & Chase (1971). For immunodiffusion 1.5 % agarose (Calbiochem) was used in 0.85’% NaCl at pH 7.2. lmmunoelectrophoresis was performed in 1.5% agarose dissolved in 0.075 Mbarbital buffer (Laurell, 1965) at pH 8.6 for 75 min at 3.5 V/cm. The two protoscolex antigens were reacted against rabbit anti-sheep serum, rabbit anti-E. grurudosus serum and against the pooled serum of sheep artificially infected with E. granulosus 2 months previously. Each of 6 sheep had approx. 50 small hydatid cysts equally distributed between lungs and livers. The two antigens were also compared with sheep serum using polyacrylamide gradient gel electrophoresis (‘Gradipore’ equipment, Townson & Mercer Pty. Ltd., Australia). Gels were scanned using an Helena Quick Scan recording densitometer after staining with Amido Black. 467

468

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D.

HEATH

and P. J.

OSE~ORN

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I.J.P. VOL. 6. 1976

Echinococcus grunukosus laminated

RESULTS During the first 14 days of culture the vesiculation of protoscoleces or the production of a posterior bladder took place as described by Smyth (1962, 1967) and Btnex (1968) (Fig. 1a, b, c). In cultures treated with pepsin for 60 min, approx. 50% of protoscoleces formed posterior bladders while 40 % vesiculated. The coalescence of posterior bladders was a feature of these cultures (Fig. lb, d, e, f). The coalesced bladders generally did not form laminated membranes unless 1O-40 protoscoleces contributed to the bladder (Fig. 2f). Such bladders appeared to possess internal structure similar to vesiculated protoscoleces (Fig. 2a) in that germinal cells lined the tegument and strands of material criss-crossed the bladder (Fig. le, f). No degeneration of protoscoleces occurred during the first 4 weeks, but during the second 4 weeks protoscoleces that had not vesiculated or formed posterior bladders began to degenerate. After 8 weeks some degeneration was also apparent in protoscoleces contributing to common bladders, although the bladders appeared normal (Fig, 2f). After 28 days of culture, membranes showing one or two laminations were present around some vesiculated protoscoleces (Fig. 2~). Laminations were more obvious in the region of the scolex (Fig. 2b, d). More vesiculated protoscoleces developed laminations between 4 and 8 weeks of culture, and the laminations became more numerous. At 8 weeks, up to 8 laminations could be seen (Fig. 2e). The rate of development of vesiculated protoscoleces in culture was similar to that in mice (Heath, 1970). In cultures treated with pepsin for only 10 min, all protoscoleces still retained remnants of brood capsule attachment at their posterior end. Vesiculation was almost 100% in those protoscoleces able to reorganise, and only a very few small posterior vesicles were seen. In these cultures laminated membranes were observed after only 17 days. The numbers of precipitation lines shown by immunodiffusion with the various antigens and antisera are shown in Table I. These were confirmed by immunoelectrophoresis. One of the arcs against each protoscolex antigen reacting with serum from E. granulosus-infected sheep appeared to correspond to the arc 5 specific E. granuiosus antigen described by Varela-Diaz, Coltorti, Ricardes, Guisantes & Yarzabal (1974). Gradient gel electrophoresis showed that al1 contaminating sheep All photographs FIG. I a. b. c. d. e.

469

membrane

proteins were still present in the cultured protoscoleces, but at approx. 25% of their original concentration. The main contaminants appeared to be IgG and albumin. DISCUSSION The present observations indicate that the laminated membrane of E. granuiosus is initially of parasite origin and probably does not require host macromolecules for its formation. The occurrence of laminations suggest that they are produced sequentially. Kilejian & Schwabe (1971) found that galactose, galactosamine and giucosamine were prime constituents of the polysaccharide portion of the laminated membrane. There could be some

relationship in the present experiment between the number of laminations and the number of media changes, because medium NCTC 135 contains only a small quantity of hexosamines. Smyth (1967) and Brudnjak, Cvetnic & Wikerhauser (1970) found that Medium 199, which does not contain galactose or hexosamines, required extensive supplementation before laminated membranes were formed. A search of the literature has revealed no reports of the coalescence of posterior bladders to form a common cyst with peripheral protoscoleces. Brudnjak et ai. (1970) described similar cysts arising by vesiculation of the germinal membrane of ruptured brood capsules. Smyth (1967) noted that posterior bladders became sticky and clumped together but they did not coalesce. Perhaps coalescence is only possible when the culture medium is relatively free of macromolecules that might provide a coating for the posterior bladders. It appears that posterior bladder formation is evidence for damage to the protoscolex during peptic digestion because Smyth (1967) found that posterior bladder formation was usual in protoscoleces that did not develop into worms. Also, BCnex (1968) observed that posterior bladders were formed if no germinal membrane was retained by the protoscolex; miniature hydatid cysts with laminated membranes developed only from vesiculated protoscoleces, and not from those with posterior bladders. In the present experiments, careful monitoring of peptic digestion increased the proportion of protoscoleces capable of vesiculation. Coltorti & Varela-Diaz (1972) pointed out that the major contaminating proteins of sheep hydatid cyst fluid were IgG and albumin. Similar results

were made in vitro using a Leitz ‘Diavert’ inverted microscope.

Echinococcus granulosus protoscoleces

forming posterior bladders. Three days itz vitro. days. a posterior bladder in the manner described by Smyth (1962)-Z% days. two posterior bladders-14 days. two posterior bladders, showing strands of material crisscrossing the bladders-14 days. 12 posterior bladders to form a structure similar to a vesiculated protoscolex-28 days.

A vesiculated protoscolex, and two others joined by their posterior bladders-7 Enlargement of Coalescence of Coalescence of

f. Coalescence

of

D. D.

kfEhTHand P. J. OSBORN

I.J.P. VOL. 6. 1976

Echinococcus gramtlosus laminated

I.J.P. VOL. 6. 1976 TABLE

I-THE

NUMBERSOF

PRECIPITATION BANDS DETECTED

471

membrane

BY IMMLJNODIFFUSION AND IMMUNOELECTROPHORESIS

Antigens Antisera Rabbit Rabbit

Normal E. granulosus

anti-sheep anti-E. granulosus

Sheep anti-E.granlr1osu.v

4 8

4

were obtained in the present experiments with protoscoleces. The contaminating proteins were reduced to a quarter of their original level by ilz vitro culture, and the cultured antigen then produced fewer precipitin lines with anti-sheep serum. Culturing of protoscoleces in a defined medium may be useful in reducing non-specific reactions during immunodiagnosis of human echinococcosis. REFERENCES J. 1968. Consid&ations expCrimentales nouvelles sur I’evolution in vitro en milieu les larves d’Eehinococcus granulosus. Annales de Parasitologic, Humaine et ComparPe (Paris) 43: 561-572. BRUDNJAK J., CVETNI~ S. & WIKERHAUSER T. 1970. Cystic development of the protoscoleces and brood capsules of Echinococcus granu/osus in cell cultures and cell-free media. Veterinurski Arhiv (Zagreb) 40: 292-296. COLTORTI E. A. & VARELA-DIAZ V. M. 1972. IgG levels and host specificity in hydatid cyst fluid. Journal oj Parasitology 58: 753-756. HEATH D. D. 1970. The developmental biology of larval cyclophyllidean cestodes in mammals. Ph.D. Thesis. The Australian National University, Canberra, Australia. pp. 235. KAGAN I. G. 1968. A review of serological tests for the diagnosis of hydatid disease. Bulletin o.f’ the World Heulth Organisation 39: 25-27. B~NEX

All photographs

were made

FIG. 2 a. A vesiculated protoscolex-14 b. Material resembling laminated c. d. e. f.

Cultured E. granulosus

Sheep serum

2 4 4

IO 4 0

KILEJIAN A., SAUER K. & SCHWABE C. W. 1962. Hostparasite relationships in Echinococcosis. VIII. Infrared spectra and chemical composition of the hydatid cyst. Experimental Parasitology 12: 377-392. KILEJIAN A. & SCHWABE C. W. 1971. Studies on the polysaccharides of the Echinococcus granulosus cyst, with observations on a possible mechanism for laminated membrane formation. Comparative Biochemistry and Physiology 40B: 25-36. LAURELL C. B. 1965. Antigen-antibody crossed electrophoresis. Analytical Biochemistry 10: 358-361. SMYTH J. D. 1962. Studies on tapeworm physiology. X. Axenic cultivation of the hydatid organism, Echinococcus granulosus; establishment of a basic technique. Parasitology 52: 44 1457. SMYTH J. D. 1967. Studies on tapeworm physiology. Xl. I/I vitro cultivation of Echinococcus granulosus from the protoscolex to the strobilate stage. Parasitology 57: I I I-133. VARELA-DIAL V. M. & C~LTORTI E. A. 1973. The presence of host immunoglobulins in hydatid cyst membranes. Journal of’ Parasitology 59: 484488. VARELA-DIAZ V. M., COLTORTI E. A., RICARDES M. I., GUISANTES J. A. & YARZABAL L. A. 1974. The immunoelectrophoretic characterization of sheep hydatid cyst Huid antigens. American Journal of Tropical Medicine and Hygiene 23: 1092-l 096. WILLIAMS C. A. & CHASE M. W. 1971. Methods in Immunology and Immunochemistry. III. Reactions of Antibodies aYth Soluble Antigens, pp. 5 15. Academic Press. New York.

in vitro using a Leitz ‘Diavert’

inverted

microscope

days. membrane appearing to emanate from the scolex region-42 days. Early laminated membrane formation around a vesiculated protoscolex-I8 days. Laminations appear to emanate from the scolex region of a vesiculated protoscolex-35 days. Laminated membrane formation continues to take place and eight laminations can be discerned-56 days. Laminated membrane formation around a coalesced posterior vesicle. Protoscoleces arc degenerating, but the coalesced vesicle appears to remain viable-56 days.