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Demonstration of a specific echinococcus multilocularis antigen in the supernatant of in vitro maintained protoscolices
Zbl. Bakt. Hyg. A 268,416-423 (1988)
Demonstration of a Specific Echinococcus multilocularis Antigen in the Supernatant of in vitro Maintained Protoscolices HERBERT AUER, KURT HERMENTIN, and HORST ASPOCK Abteilung fiir Medizinische Parasitologie (Leiter: Univ.-Prof. Dr. H. Aspock) des HygieneInstituts der Universitat Wien (Vorstand: Univ.-Prof. Dr. H. Flamm)
With 4 Figures' Received June 25, 1987 . Accepted September 28, 1987
Abstract Serodiagnosis of echinococcosis is still often met with difficulties resulting from unspecific reactions due to crude antigens which contain numerous host-derived proteins. In order to eliminate host protein contamination we produced Echinococcus multilocularis antigen by methods of in vitro technique: Evaginated protoscolices of Echinococcus multilocularis isolated from experimentally infected Mongolian gerbils (Meriones unguiculatus) were maintained in RPMI 1640 medium. Although no serum proteins were added, protoscolices could be kept alive for more than 2 weeks. The supernatants harvested from protoscolices cultures were tested for their immunoreactivity against sera of patients with confirmed alveolar or cystic echinococcosis, cysticercosis, schistosomiasis or fascioliasis by means of SDS-PAGE and immunoblotting. A polypeptide band at about 62,000 mol. mass was identified which proved to be specifically immunoreactive with sera from patients with alveolar echinococcosis, whereas sera from patients with cystic echinococcosis or with other helminthic infections as well as sera from healthy blood-donors did not react with this polypeptide band. Thus, the use of supernatant antigen in immunoblotting technique allows a serological differentiation between infections with Echinococcus multilocularis and those with Echinococcus granulosus and provides an accurate diagnosis of alveolar echinococcosis.
Zusammenfassung Es wird ein in vitro-Modell zur Haltung von Proto scolices von Echinococcus multilocularis in serumfreiem Medium vorgestellt und iiber die Isolierung eines E. multilocularis-spezifischen Antigens aus den wahrend der in vitro-Haltung gewonnenen Kultuniberstanden berichtet. Proto scolices wurden aus dem Metazestodengewebe experimentell infizierter Wiistenrennmiiuse (Meriones unguiculatus) isoliert und in Gewebekulturflaschen mit RPMI 1640-Medium transferiert. Die evaginierten Proto scolices konnten im serumfreien Medium unter definierten Bedingungen mehr als zwei Wochen am Leben gehalten werden. Die wahrend der in vitro-Haltung gewonnenen, an Stoffwechselprodukten der Proto scolices reichen Kulnmibersrande wurden gesammelt und in der SDS-PAGE aufgetrennt. Im Anschluf daran wurde die Imrnunreaktivitat der aufgetrennten Polypeptidbanden in einem Immunoblot-Verfahren getestet. Eine Polypeptidbande (MW ca. 62.000) er-
In vitro Production of Echinococcus multilocularis Antigen
417
wies sich dabei als E. multilocularis-spezifisch: Sie reagierte ausschliefslich mit Seren von Patienten mit alveolarer Echinokokkose, nicht aber mit Seren von Patienten mit zystischer
Echinokokkose, mit Fasziolose, Zystizerkose oder Bilharziose, oder mit Seren von gesunden Blutspendern. Die Verwendung von Uberstandsantigen in einem Immunoblot-Verfahren errnoglicht eine sichere und artspezifische Serodiagnose der alveolaren Echinokokkose.
Introduction Alveolar echinococcosis caused by metacestodes of Echinococcus multilocularis is one of the most dangerous helminthic infections in man. It is characterized by an extraordinarily severe and chronic course. Surgical resection of the entire primary hepatic lesion offers the only proven curative treatment; however, at the time of diagnosis the disease often has progressed far so that total resection is impossible. Thus, an early and accurate diagnosis of alveolar echinococcosis is important for the therapeutical procedure and consequently for the prognosis of the disease. The diagnosis of alveolar echinococcosis is mainly based on the detection of specific antibodies by highly sensitive serological tests. The specificity and consequently the significance of these tests depend on the purity of the antigen used: Crude antigens prepared from homogenized metacestodes of Echinococcus multilocularis or E. granulosus respectively consist of undefined complexes of numerous antigens and contain high quantities of host proteins causing unspecific reactions in serological tests (4, 11, 16). The purification of such crude antigens by biochemical and immunochemical methods leads to an essential decrease of interfering reactions (5, 7, 8, 10). However, these purified preparations still contain host proteins (6). In contrast to the conventional strategy for the production of Echinococcus antigen (i.e. purification of crude antigens) we intended to prepare pure antigen. The present paper reports on the production of E. multilocularis antigen obtained from supernatants of in vitro maintained protoscolices. The supernatant antigens were analysed by SDS-PAGE and the immunoreactivity of separated polypeptides was revealed by immunoblotting.
Materials and Methods
In vivo maintenance of E. multilocularis Metacestodes of E. multilocularis were maintained in Mongolian gerbils (Meriones unguiculatus) by intraperitoneal serial passage of protoscolices. For the production of supernatant antigen metacestode material of E. multilocularis (strain S 81 isolated in Southern Germany) was obtained from the peritoneal cavity of a gerbil infected four months previously.
Isolation of protoscolices Metacestode material was removed from the peritoneal cavity asepticallyaccording to the methods of Smyth and Davies (13, 14). After careful dissection the metacestodes were washed several times in prewarmed Hanks' saline (Flow Laboratories, GB) containing 1% antibiotics and antimycotics + 1% neomycine(Gibco, GB).With the aid of a glasspestlethe parasite mass was successively pressed through metal mesh screens (EO.B., USA) of 230 urn
418
H. Auer, K. Hermentin, and H. Aspock
pore size. After several washing steps in Hanks' saline, followed by repeated filtration procedures through a 180 urn pore size screen a pure suspension of protoscolices free of flocculent tissue debris could be obtained. Prior to transfer into culture vesselsprotoscolices were checked for viability (motility + flame cell activity).
In vitro maintenance of protoscolices Four separate cultures were set up in 25 ml polystyrol culture vessels (Costar, USA) with RPMI 1640 medium (Gibco) supplemented with 1% antibiotics and antimycotics +1% neomycine. A total of approximately 25,000 protoscolices per flask were incubated in 10 ml Culture vessels were agitated by hand several medium (gas phase: 100% air) at + times a day. The medium was renewed every 24 h on day 1 and 2 and every 48 h from day 3 until day 19 (after transfer into culture flasks protoscolices were subjected to repeated washing procedures with Hank's saline on day 1 and 2 before adding fresh medium). Prior to renewing the medium the four cultures were checked for the presence of microorganisms by microscopical examination, and the viability (motility + flame cell activity) of protoscolices was examined. The proportion of viable protoscolices per culture (in %) was expressed as mean value calculated after examining 100 proto scolices per flask.
3rc.
Preparation of supernatant antigen Supernatants harvested from cultures were filtered through a Millex GS 0.22 um filter (Millipore, France) and immediately stored at -20°C. For antigen preparation the supernatants of days 3, 5, 7, 9, 11 and 13 were thawed and pooled whereas the supernatants of day 1 and 2 as well as those of day 15 and later were discarded. The pooled supernatants were tenfold concentrated by polyethylene glycol 20,000 (Merck, FRG), dialysed against PBS pH 7.2 at + 4°C for 48 h and finally precipitated by trichloroacetic acid (Merck).
SDS-PAGE analysis of supernatant antigen Electrophoresis under reducing conditions was performed on a GE 2/4 LS vertical electrophoresis unit (Pharmacia Fine Chemicals, Sweden) using the discontinuous buffer system of Laemmli (9) with a stacking gel of 6% and a separating gel of 12.5% acrylamide/ bisacrylamide (BioRad, USA). Molecular standards were purchased from BioRad. Samples (protein content: 35 ug) for analysis were boiled for 5 min in Laemmli sample buffer containing 2% SDS, 5% mercaptoethanol and 10% glycerol. Gels were electrophoresed at 150 V constant voltage for 5 h at + 15°C and thereafter stained with 0.2% Coomassie blue.
Immunological analysis of supernatant antigen by immunoblotting Supernatant antigen previously separated by SDS-PAGE was electrophoretically transferred (35 V and 100 rnA) for 18 h from separating gel onto nitrocellulose membrane (BioRad) according to the method of Towbin et al. (15). After transfer the membranes were cut into strips and coated in PBS containing 5% BSA. The coated strips were treated with 2 ml serum dilution (1:100 in PBS + 5% BSA) for one hour at room temperature. After removal of unbound antibodies by washing in PBS, the strips were incubated with 2 ml of peroxidase-labelled goat anti-human IgG or goat antirabbit IgG, respectively (both conjugates from Cappel Laboratories, USA;conjugate dilution 1 : 500 in PBS + 5% BSA) for 1 h at room temperature. Excess of conjugate was removed by a five-step-wash with PBS and finally the chloronaphthol substrate was added.
Sera - 20 human sera were obtained from patients with proven alveolar echinococcosis and 20 human sera from patients with cystic echinococcosis. The infections were confirmed by computed tomography/sonography, surgery or histology.
In vitro Production of Echinococcus multilocularis Antigen
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- 2 human sera were obtained from patients with clinically and parasitologically confirmed cysticercosis, 2 sera from patients with fascioliasis and 1 serum from a patient with schistosomiasis. - 2 human sera were obtained from healthy blood-donors (normal human sera). - 1 rabbit anti-mouse serum (DAKO, Denmark) was used to check supernatant antigen for the presence of mouse proteins.
Results
In vitro maintenance of protoscolices Most protoscolices isolated from larval cyst mass evaginated in Hanks's saline during washing procedures. 24 h after transfer into RPMI 1640 medium about 95 % of freed protoscolices were evaginated (Fig. 1). Organisms showed a good degree of motility from the onset of maintenance in all cultures. Protoscolices could be kept alive in serum-free RPMI 1640 medium for at least 15 days (Fig. 2). From day 15 onwards the number of active protoscolices rapidly decreased, and finally on day 21 nearly no flame cell activity could be observed. The morphological development of protoscolices during in vitro maintenance was characterized by a small increase of body size, the appearance of the outlines of the secretory canals and disappearance of the calcareous corpuscles. After the 15 th day of maintenance, the movements of the protoscolices ceased and the hooks became detached from the rostellum; this process was quickly followed by degeneration, vesiculation and disintegration.
SDS-PAGE analysis and immunoblotting Fig. 3 illustrates the polypeptide pattern of the supernatant antigen analysed by SDSPAGE. The most prominent polypeptide band could be observed between 65,000 and 62,000 in mol. mass. Five less distinct polypeptide bands were recognizable at about 60,000, 57,000, 55,000, 52,000 and 47,000 daltons. In order to check supernatant antigen for immunoreactive components the electrophoresed polypeptides were transferred onto nitrocellulose membranes. The irnmunoblotting analysis of human anti-E. multilocularis and anti-E. granulosus sera is demonstrated in Fig. 4. Each of the E. multilocularis sera (n = 20) revealed four bands; one very strong immunoreaction at 62,000, weak reactions at 60,000, 57,000 and 55,000 mol. mass while E. granulosus sera (n = 20) were directed against polypeptides with estimated mol. masses at 60,000 and 57,000 (weak reactions). The polypeptide band at about 62,000 mol. mass proved to be specifically immunoreactive with sera from patients with alveolar echinococcosis. Normal human sera as well as sera of patients infected with various other helminths did not show any visible reactions (bands) after immunostaining.
Discussion In contrast to the conventional strategy for the production and preparation of
Echinococcus antigen, i.e, biochemical and/or immunochemical purification of homogenized metacestodes of E. multilocularis or E. granulosus respectively, we tried to produce pure E. multilocularis antigen by an in vitro technique: Proto scolices of a 26 Zbl. Bakt. H yg. A 268/3
H. Auer, K. Hermentin, and H. Aspock
420
,,
Fig. 1. Evaginated protoscolex of E. multilocularis in serum-free RPMI 1640 medium 48 h after onset of the culture (magnification, x 400).
100
c o (; a
o
Q
Fig. 2. In vitro maintenance of proto scolices of E. multilocularis in serum-free RPMI medium: Proportion of viable protoscolices in four separate cultures (0-0, A - A , .....- ....., __) examined prior to renewing the medium.
In vitro Production of Echinococcus multilocularis Antigen
421
92 ,5 ~ 66 ,2 ~
45 ~
45 •
31 ~
31 •
2 1,5 ~
.
v •
Fig. 3. Coomassie stained SDS-PAGE pattern of E. multilocularis supernatant antigen. Molecular weight standards are indicated in kilodaltons. Fig. 4. Immunoblot of supernatant antigen tested with a human anti-E. multilocularis (lane A) or anti-E. granulosus serum (lane B). Molecular weight standards are indicated in kilodaltons.
highly fertile E. multilocularis isolate were taken from an experimentally infected laboratory animal and transferred into culture flasks where they were maintained under defined conditions in a serum-free tissue culture medium, supplemented only with antibiotics and antimycotics. The supernatants harvested from these cultures were checked for the presence of immunoreactive components by SDS-PAGE and imrnunoblotting. In vitro maintenance of protoscolices of E. multilocularis (isolate S 81) could be performed in serum-free RPMI 1640 medium - a variety of different tissue culture
422
H. Auer, K.Hermentin, and H. Aspock
media had been tested previously (2, 3) - throughout more than two weeks. Most protoscolices evaginated prior to the transfer into culture vessels without any treatment with evagination solution (14); this is probably due to the isolation procedure (mechanical pressure) and the change of pH conditions during the washing steps. During in vitro maintenance no essential morphological development - as it can be observed during cultivation of E. multilocularis strobilar stages (14) - could be realized; they only developed to presegmental stage 2 according to the development scheme of Smyth (12). After day 15 of maintenance, protoscolices began to vesiculate and to decay, and finally on day 21 nearly all protoscolices were disintegrated. SDS-PAGE and immunoblotting analysis were carried out with supernatants harvested between day 3 and 13, when protoscolices showed high motility and intensive flame cell activity. In order to eliminate host proteins possibly transferred into culture medium at the beginning of in vitro maintenance, protoscolices were subjected to several washing procedures on day 1 and 2, and supernatants harvested on these two days were discarded. Supernatants of day 15 and thereafter were also discarded to exclude an accumulation of somatic antigens deriving from degenerating protoscolices. In order to exclusively use the protein components of supernatant antigen for SDSPAGE analysis and immunoblotting, TCA precipitation was carried out. Although some antigenic reactivity of the polypeptides might have been destroyed by TCA and although electrophoretic separation was carried through under reducing conditions, several polypeptide bands could be detected by anti-E. multilocularis and anti-E. granulosus sera when blotted onto nitrocellulose membranes. Sera from patients with confirmed cysticercosis,schistosomiasis or fascioliasis,which very often show serological cross-reactions with Echinococcus-antigen (1) did not react with any of these polypeptides. The presence of host-deriving proteins in the supernatants was checked by the use of a polyclonal anti-mouse serum in an immunoblot test. In spite of the fact that mice and gerbils are different animal species their serum protein pattern showed identical bands in immunoblot using an anti-mouse serum (author's experience, unpublished), and thus it was used to check on the presence of host proteins. No reactions of the separated supernatant antigen with the anti-mouse serum were detectable by SDS-PAGE/ immunoblot analysis. When comparing the immunoblots a clear differentiation between sera of patients with alveolar and cystic echinococcosis was possible: The polypeptide at 62,000 mol. mass reacted in immunoblot clearly and exclusively with antibodies of patients with alveolar echinococcosis. This demonstrates that the 62,000 mol. mass polypeptide is specific to E. multilocularis, thus allowing a species-specific differentiation. The polypeptide presumable is part of an antigen released by the protoscolices into the culture medium (excretory/secretory antigen) in a period of high activity and metabolism. A specific E. multilocularis antigen (at 54,000 mol. mass) which likewise was described to allow a species-specific differentiation has been isolated by Gottstein (6) from crude antigen by affinity chromatography. Whether or not these antigens are of (partially) identical origin remains to be examined. The use of supernatant antigen produced in vitro under serum-free defined conditions in immunoblotting technique, as described in the present paper, may be considered as a promising tool for an accurate and definite diagnosis of alveolar echinococcosis. This is of utmost interest in the early diagnosis of echinococcosis particularly in cases of asymptomatic infections in areas where both E. multilocularis and E. granulosus occur.
In vitro Production of Echinococcus multilocularis Antigen
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Acknowledgements. The authors would like to thank Prof. Dr. H. Flamm for his invaluable help in providing laboratory facilities. We are indebted to Miss M. Klaudus for her excellent technical assistance. We also gratefully acknowledge the support by Janssen Pharmaceutica GmbH, Vienna, Austria.
References
1. Ambroise-Thomas, P. and P. T. Desgorges: Valeur diagnostique et limites du test ELISA applique
a l'hydatidose. Bull. Soc. Path. Exot.
73 (1980) 89-99
2. Auer,H. and H. Aspock: Studies on antigens from in vitro cultivated protoscolices of Echinococcus multilocularis and their possible use in the serodiagnosis of human echinococccosis. Proc. 2nd Symp. Taeniasis/Cysticercosis and Echinococcosis/Hydatidosis, Dec. 1985, Ceske Budejovice, CSSR (1986) 7-15 3. Auer, H., K. Hermentin, and H. Aspoci« In vitro-Haltung von Protoscolices von Echinococus multilocularis. Mitt. Osterr. Ges. Tropenmed. Parasitol. 8 (1986) 99-103 4. Ben Ismail, R. B., B. Carme, G. Niel, and M. Gentilini: Nonspecific serological reactions with Echinococcus granulosus antigens: role of anti-P 1 antibodies. Am. J. Trop. Med. Hyg. 29 (1980) 239-245 5. Farag, H., D. Bout, and A. Capron: Specific immunodiagnosis of hydatidosis by enzyme-linked immunosorbent asssay (ELISA). Biomedecine 23 (1975) 276-278 6. Gottstein, B.: Purification and characterization of a specific antigen from Echinococcus multilocularis. Parasite Immunol. 7 (1985) 201-212 7. Gottstein, B., ]. Eckert, and H. Fey: Serological differentiation between Echinococcus granulosus and E. multilocularis infections in man. Z. Parasitenkd. 69 (1983) 347-356 8. Hess, V.,]. Eckert, and A. Frohlich: Vergleich serologischer Methoden fur die Diagnose der zystischen und alveolaren Echinokokkose des Menschen. Schweiz. med. Wschr. 104 (1974) 853-859 9. Laemmli,]. H.: Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227 (1970) 680-685 10. Merioua, A., D. Bout, and A. Capron: Evaluation of ELISA and RAST using purified antigens for diagnosis of hydatidosis. J. Path. Biol, 32 (1984) 15-22 11. Schantz, P. M., D. Shanks, and M. Wilson: Serologic cross-reactions with sera from patients with echinococcosis and cysticercosis. Am. J. Trop. Med. Hyg. 29 (1980) 609-612 12. Smyth, ]. D.: Echinococcus granulosus and E. multilocularis: In vitro culture of the strobilar stages from protoscoleces. Angew. Parasit. 20 (1979) 137-147 13. Smyth, ]. D. and Z. Davies: In vitro culture of strobilar stages in Echinococcus granulosus (sheep strain): A review of basic poblems and results. Int. J. Parasitol. 4 (1974) 631-44 14. Smyth, J. D. and Z. Davies: In vitro suppression of segmentation in Echinococcus multilocularis with morphological transformation of protoscoleces into mono zoic adults. Parasitology 71 (1975) 125-135 15. Towbin, H., T. Staehlin, and]. Gordon: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Nat. Acad. Sci. USA 76 (1979) 4350-4354 16. Varela-Diaz, V. M.,]. A. Guisantes, M.]. Ricardes, L. A. Yarzabal, and E. A. Coltorti: Evaluation of whole and purified hydatid fluid antigens in the diagnosis of human hydatidosis by the immunoelectrophoresis test. Am. J. Trop. Med. Hyg. 24 (1975) 298-303 Dr. H. Auer, Department of Med. Parasitology, Institute of Hygiene, Kinderspitalgasse 15, A-1094 Vienna, Austria.
Demonstration of a Specific Echinococcus multilocularis Antigen in the Supernatant of in vitro Maintained Protoscolices HERBERT AUER, KURT HERMENTIN, and HORST ASPOCK Abteilung fiir Medizinische Parasitologie (Leiter: Univ.-Prof. Dr. H. Aspock) des HygieneInstituts der Universitat Wien (Vorstand: Univ.-Prof. Dr. H. Flamm)
With 4 Figures' Received June 25, 1987 . Accepted September 28, 1987
Abstract Serodiagnosis of echinococcosis is still often met with difficulties resulting from unspecific reactions due to crude antigens which contain numerous host-derived proteins. In order to eliminate host protein contamination we produced Echinococcus multilocularis antigen by methods of in vitro technique: Evaginated protoscolices of Echinococcus multilocularis isolated from experimentally infected Mongolian gerbils (Meriones unguiculatus) were maintained in RPMI 1640 medium. Although no serum proteins were added, protoscolices could be kept alive for more than 2 weeks. The supernatants harvested from protoscolices cultures were tested for their immunoreactivity against sera of patients with confirmed alveolar or cystic echinococcosis, cysticercosis, schistosomiasis or fascioliasis by means of SDS-PAGE and immunoblotting. A polypeptide band at about 62,000 mol. mass was identified which proved to be specifically immunoreactive with sera from patients with alveolar echinococcosis, whereas sera from patients with cystic echinococcosis or with other helminthic infections as well as sera from healthy blood-donors did not react with this polypeptide band. Thus, the use of supernatant antigen in immunoblotting technique allows a serological differentiation between infections with Echinococcus multilocularis and those with Echinococcus granulosus and provides an accurate diagnosis of alveolar echinococcosis.
Zusammenfassung Es wird ein in vitro-Modell zur Haltung von Proto scolices von Echinococcus multilocularis in serumfreiem Medium vorgestellt und iiber die Isolierung eines E. multilocularis-spezifischen Antigens aus den wahrend der in vitro-Haltung gewonnenen Kultuniberstanden berichtet. Proto scolices wurden aus dem Metazestodengewebe experimentell infizierter Wiistenrennmiiuse (Meriones unguiculatus) isoliert und in Gewebekulturflaschen mit RPMI 1640-Medium transferiert. Die evaginierten Proto scolices konnten im serumfreien Medium unter definierten Bedingungen mehr als zwei Wochen am Leben gehalten werden. Die wahrend der in vitro-Haltung gewonnenen, an Stoffwechselprodukten der Proto scolices reichen Kulnmibersrande wurden gesammelt und in der SDS-PAGE aufgetrennt. Im Anschluf daran wurde die Imrnunreaktivitat der aufgetrennten Polypeptidbanden in einem Immunoblot-Verfahren getestet. Eine Polypeptidbande (MW ca. 62.000) er-
In vitro Production of Echinococcus multilocularis Antigen
417
wies sich dabei als E. multilocularis-spezifisch: Sie reagierte ausschliefslich mit Seren von Patienten mit alveolarer Echinokokkose, nicht aber mit Seren von Patienten mit zystischer
Echinokokkose, mit Fasziolose, Zystizerkose oder Bilharziose, oder mit Seren von gesunden Blutspendern. Die Verwendung von Uberstandsantigen in einem Immunoblot-Verfahren errnoglicht eine sichere und artspezifische Serodiagnose der alveolaren Echinokokkose.
Introduction Alveolar echinococcosis caused by metacestodes of Echinococcus multilocularis is one of the most dangerous helminthic infections in man. It is characterized by an extraordinarily severe and chronic course. Surgical resection of the entire primary hepatic lesion offers the only proven curative treatment; however, at the time of diagnosis the disease often has progressed far so that total resection is impossible. Thus, an early and accurate diagnosis of alveolar echinococcosis is important for the therapeutical procedure and consequently for the prognosis of the disease. The diagnosis of alveolar echinococcosis is mainly based on the detection of specific antibodies by highly sensitive serological tests. The specificity and consequently the significance of these tests depend on the purity of the antigen used: Crude antigens prepared from homogenized metacestodes of Echinococcus multilocularis or E. granulosus respectively consist of undefined complexes of numerous antigens and contain high quantities of host proteins causing unspecific reactions in serological tests (4, 11, 16). The purification of such crude antigens by biochemical and immunochemical methods leads to an essential decrease of interfering reactions (5, 7, 8, 10). However, these purified preparations still contain host proteins (6). In contrast to the conventional strategy for the production of Echinococcus antigen (i.e. purification of crude antigens) we intended to prepare pure antigen. The present paper reports on the production of E. multilocularis antigen obtained from supernatants of in vitro maintained protoscolices. The supernatant antigens were analysed by SDS-PAGE and the immunoreactivity of separated polypeptides was revealed by immunoblotting.
Materials and Methods
In vivo maintenance of E. multilocularis Metacestodes of E. multilocularis were maintained in Mongolian gerbils (Meriones unguiculatus) by intraperitoneal serial passage of protoscolices. For the production of supernatant antigen metacestode material of E. multilocularis (strain S 81 isolated in Southern Germany) was obtained from the peritoneal cavity of a gerbil infected four months previously.
Isolation of protoscolices Metacestode material was removed from the peritoneal cavity asepticallyaccording to the methods of Smyth and Davies (13, 14). After careful dissection the metacestodes were washed several times in prewarmed Hanks' saline (Flow Laboratories, GB) containing 1% antibiotics and antimycotics + 1% neomycine(Gibco, GB).With the aid of a glasspestlethe parasite mass was successively pressed through metal mesh screens (EO.B., USA) of 230 urn
418
H. Auer, K. Hermentin, and H. Aspock
pore size. After several washing steps in Hanks' saline, followed by repeated filtration procedures through a 180 urn pore size screen a pure suspension of protoscolices free of flocculent tissue debris could be obtained. Prior to transfer into culture vesselsprotoscolices were checked for viability (motility + flame cell activity).
In vitro maintenance of protoscolices Four separate cultures were set up in 25 ml polystyrol culture vessels (Costar, USA) with RPMI 1640 medium (Gibco) supplemented with 1% antibiotics and antimycotics +1% neomycine. A total of approximately 25,000 protoscolices per flask were incubated in 10 ml Culture vessels were agitated by hand several medium (gas phase: 100% air) at + times a day. The medium was renewed every 24 h on day 1 and 2 and every 48 h from day 3 until day 19 (after transfer into culture flasks protoscolices were subjected to repeated washing procedures with Hank's saline on day 1 and 2 before adding fresh medium). Prior to renewing the medium the four cultures were checked for the presence of microorganisms by microscopical examination, and the viability (motility + flame cell activity) of protoscolices was examined. The proportion of viable protoscolices per culture (in %) was expressed as mean value calculated after examining 100 proto scolices per flask.
3rc.
Preparation of supernatant antigen Supernatants harvested from cultures were filtered through a Millex GS 0.22 um filter (Millipore, France) and immediately stored at -20°C. For antigen preparation the supernatants of days 3, 5, 7, 9, 11 and 13 were thawed and pooled whereas the supernatants of day 1 and 2 as well as those of day 15 and later were discarded. The pooled supernatants were tenfold concentrated by polyethylene glycol 20,000 (Merck, FRG), dialysed against PBS pH 7.2 at + 4°C for 48 h and finally precipitated by trichloroacetic acid (Merck).
SDS-PAGE analysis of supernatant antigen Electrophoresis under reducing conditions was performed on a GE 2/4 LS vertical electrophoresis unit (Pharmacia Fine Chemicals, Sweden) using the discontinuous buffer system of Laemmli (9) with a stacking gel of 6% and a separating gel of 12.5% acrylamide/ bisacrylamide (BioRad, USA). Molecular standards were purchased from BioRad. Samples (protein content: 35 ug) for analysis were boiled for 5 min in Laemmli sample buffer containing 2% SDS, 5% mercaptoethanol and 10% glycerol. Gels were electrophoresed at 150 V constant voltage for 5 h at + 15°C and thereafter stained with 0.2% Coomassie blue.
Immunological analysis of supernatant antigen by immunoblotting Supernatant antigen previously separated by SDS-PAGE was electrophoretically transferred (35 V and 100 rnA) for 18 h from separating gel onto nitrocellulose membrane (BioRad) according to the method of Towbin et al. (15). After transfer the membranes were cut into strips and coated in PBS containing 5% BSA. The coated strips were treated with 2 ml serum dilution (1:100 in PBS + 5% BSA) for one hour at room temperature. After removal of unbound antibodies by washing in PBS, the strips were incubated with 2 ml of peroxidase-labelled goat anti-human IgG or goat antirabbit IgG, respectively (both conjugates from Cappel Laboratories, USA;conjugate dilution 1 : 500 in PBS + 5% BSA) for 1 h at room temperature. Excess of conjugate was removed by a five-step-wash with PBS and finally the chloronaphthol substrate was added.
Sera - 20 human sera were obtained from patients with proven alveolar echinococcosis and 20 human sera from patients with cystic echinococcosis. The infections were confirmed by computed tomography/sonography, surgery or histology.
In vitro Production of Echinococcus multilocularis Antigen
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- 2 human sera were obtained from patients with clinically and parasitologically confirmed cysticercosis, 2 sera from patients with fascioliasis and 1 serum from a patient with schistosomiasis. - 2 human sera were obtained from healthy blood-donors (normal human sera). - 1 rabbit anti-mouse serum (DAKO, Denmark) was used to check supernatant antigen for the presence of mouse proteins.
Results
In vitro maintenance of protoscolices Most protoscolices isolated from larval cyst mass evaginated in Hanks's saline during washing procedures. 24 h after transfer into RPMI 1640 medium about 95 % of freed protoscolices were evaginated (Fig. 1). Organisms showed a good degree of motility from the onset of maintenance in all cultures. Protoscolices could be kept alive in serum-free RPMI 1640 medium for at least 15 days (Fig. 2). From day 15 onwards the number of active protoscolices rapidly decreased, and finally on day 21 nearly no flame cell activity could be observed. The morphological development of protoscolices during in vitro maintenance was characterized by a small increase of body size, the appearance of the outlines of the secretory canals and disappearance of the calcareous corpuscles. After the 15 th day of maintenance, the movements of the protoscolices ceased and the hooks became detached from the rostellum; this process was quickly followed by degeneration, vesiculation and disintegration.
SDS-PAGE analysis and immunoblotting Fig. 3 illustrates the polypeptide pattern of the supernatant antigen analysed by SDSPAGE. The most prominent polypeptide band could be observed between 65,000 and 62,000 in mol. mass. Five less distinct polypeptide bands were recognizable at about 60,000, 57,000, 55,000, 52,000 and 47,000 daltons. In order to check supernatant antigen for immunoreactive components the electrophoresed polypeptides were transferred onto nitrocellulose membranes. The irnmunoblotting analysis of human anti-E. multilocularis and anti-E. granulosus sera is demonstrated in Fig. 4. Each of the E. multilocularis sera (n = 20) revealed four bands; one very strong immunoreaction at 62,000, weak reactions at 60,000, 57,000 and 55,000 mol. mass while E. granulosus sera (n = 20) were directed against polypeptides with estimated mol. masses at 60,000 and 57,000 (weak reactions). The polypeptide band at about 62,000 mol. mass proved to be specifically immunoreactive with sera from patients with alveolar echinococcosis. Normal human sera as well as sera of patients infected with various other helminths did not show any visible reactions (bands) after immunostaining.
Discussion In contrast to the conventional strategy for the production and preparation of
Echinococcus antigen, i.e, biochemical and/or immunochemical purification of homogenized metacestodes of E. multilocularis or E. granulosus respectively, we tried to produce pure E. multilocularis antigen by an in vitro technique: Proto scolices of a 26 Zbl. Bakt. H yg. A 268/3
H. Auer, K. Hermentin, and H. Aspock
420
,,
Fig. 1. Evaginated protoscolex of E. multilocularis in serum-free RPMI 1640 medium 48 h after onset of the culture (magnification, x 400).
100
c o (; a
o
Q
Fig. 2. In vitro maintenance of proto scolices of E. multilocularis in serum-free RPMI medium: Proportion of viable protoscolices in four separate cultures (0-0, A - A , .....- ....., __) examined prior to renewing the medium.
In vitro Production of Echinococcus multilocularis Antigen
421
92 ,5 ~ 66 ,2 ~
45 ~
45 •
31 ~
31 •
2 1,5 ~
.
v •
Fig. 3. Coomassie stained SDS-PAGE pattern of E. multilocularis supernatant antigen. Molecular weight standards are indicated in kilodaltons. Fig. 4. Immunoblot of supernatant antigen tested with a human anti-E. multilocularis (lane A) or anti-E. granulosus serum (lane B). Molecular weight standards are indicated in kilodaltons.
highly fertile E. multilocularis isolate were taken from an experimentally infected laboratory animal and transferred into culture flasks where they were maintained under defined conditions in a serum-free tissue culture medium, supplemented only with antibiotics and antimycotics. The supernatants harvested from these cultures were checked for the presence of immunoreactive components by SDS-PAGE and imrnunoblotting. In vitro maintenance of protoscolices of E. multilocularis (isolate S 81) could be performed in serum-free RPMI 1640 medium - a variety of different tissue culture
422
H. Auer, K.Hermentin, and H. Aspock
media had been tested previously (2, 3) - throughout more than two weeks. Most protoscolices evaginated prior to the transfer into culture vessels without any treatment with evagination solution (14); this is probably due to the isolation procedure (mechanical pressure) and the change of pH conditions during the washing steps. During in vitro maintenance no essential morphological development - as it can be observed during cultivation of E. multilocularis strobilar stages (14) - could be realized; they only developed to presegmental stage 2 according to the development scheme of Smyth (12). After day 15 of maintenance, protoscolices began to vesiculate and to decay, and finally on day 21 nearly all protoscolices were disintegrated. SDS-PAGE and immunoblotting analysis were carried out with supernatants harvested between day 3 and 13, when protoscolices showed high motility and intensive flame cell activity. In order to eliminate host proteins possibly transferred into culture medium at the beginning of in vitro maintenance, protoscolices were subjected to several washing procedures on day 1 and 2, and supernatants harvested on these two days were discarded. Supernatants of day 15 and thereafter were also discarded to exclude an accumulation of somatic antigens deriving from degenerating protoscolices. In order to exclusively use the protein components of supernatant antigen for SDSPAGE analysis and immunoblotting, TCA precipitation was carried out. Although some antigenic reactivity of the polypeptides might have been destroyed by TCA and although electrophoretic separation was carried through under reducing conditions, several polypeptide bands could be detected by anti-E. multilocularis and anti-E. granulosus sera when blotted onto nitrocellulose membranes. Sera from patients with confirmed cysticercosis,schistosomiasis or fascioliasis,which very often show serological cross-reactions with Echinococcus-antigen (1) did not react with any of these polypeptides. The presence of host-deriving proteins in the supernatants was checked by the use of a polyclonal anti-mouse serum in an immunoblot test. In spite of the fact that mice and gerbils are different animal species their serum protein pattern showed identical bands in immunoblot using an anti-mouse serum (author's experience, unpublished), and thus it was used to check on the presence of host proteins. No reactions of the separated supernatant antigen with the anti-mouse serum were detectable by SDS-PAGE/ immunoblot analysis. When comparing the immunoblots a clear differentiation between sera of patients with alveolar and cystic echinococcosis was possible: The polypeptide at 62,000 mol. mass reacted in immunoblot clearly and exclusively with antibodies of patients with alveolar echinococcosis. This demonstrates that the 62,000 mol. mass polypeptide is specific to E. multilocularis, thus allowing a species-specific differentiation. The polypeptide presumable is part of an antigen released by the protoscolices into the culture medium (excretory/secretory antigen) in a period of high activity and metabolism. A specific E. multilocularis antigen (at 54,000 mol. mass) which likewise was described to allow a species-specific differentiation has been isolated by Gottstein (6) from crude antigen by affinity chromatography. Whether or not these antigens are of (partially) identical origin remains to be examined. The use of supernatant antigen produced in vitro under serum-free defined conditions in immunoblotting technique, as described in the present paper, may be considered as a promising tool for an accurate and definite diagnosis of alveolar echinococcosis. This is of utmost interest in the early diagnosis of echinococcosis particularly in cases of asymptomatic infections in areas where both E. multilocularis and E. granulosus occur.
In vitro Production of Echinococcus multilocularis Antigen
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Acknowledgements. The authors would like to thank Prof. Dr. H. Flamm for his invaluable help in providing laboratory facilities. We are indebted to Miss M. Klaudus for her excellent technical assistance. We also gratefully acknowledge the support by Janssen Pharmaceutica GmbH, Vienna, Austria.
References
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2. Auer,H. and H. Aspock: Studies on antigens from in vitro cultivated protoscolices of Echinococcus multilocularis and their possible use in the serodiagnosis of human echinococccosis. Proc. 2nd Symp. Taeniasis/Cysticercosis and Echinococcosis/Hydatidosis, Dec. 1985, Ceske Budejovice, CSSR (1986) 7-15 3. Auer, H., K. Hermentin, and H. Aspoci« In vitro-Haltung von Protoscolices von Echinococus multilocularis. Mitt. Osterr. Ges. Tropenmed. Parasitol. 8 (1986) 99-103 4. Ben Ismail, R. B., B. Carme, G. Niel, and M. Gentilini: Nonspecific serological reactions with Echinococcus granulosus antigens: role of anti-P 1 antibodies. Am. J. Trop. Med. Hyg. 29 (1980) 239-245 5. Farag, H., D. Bout, and A. Capron: Specific immunodiagnosis of hydatidosis by enzyme-linked immunosorbent asssay (ELISA). Biomedecine 23 (1975) 276-278 6. Gottstein, B.: Purification and characterization of a specific antigen from Echinococcus multilocularis. Parasite Immunol. 7 (1985) 201-212 7. Gottstein, B., ]. Eckert, and H. Fey: Serological differentiation between Echinococcus granulosus and E. multilocularis infections in man. Z. Parasitenkd. 69 (1983) 347-356 8. Hess, V.,]. Eckert, and A. Frohlich: Vergleich serologischer Methoden fur die Diagnose der zystischen und alveolaren Echinokokkose des Menschen. Schweiz. med. Wschr. 104 (1974) 853-859 9. Laemmli,]. H.: Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227 (1970) 680-685 10. Merioua, A., D. Bout, and A. Capron: Evaluation of ELISA and RAST using purified antigens for diagnosis of hydatidosis. J. Path. Biol, 32 (1984) 15-22 11. Schantz, P. M., D. Shanks, and M. Wilson: Serologic cross-reactions with sera from patients with echinococcosis and cysticercosis. Am. J. Trop. Med. Hyg. 29 (1980) 609-612 12. Smyth, ]. D.: Echinococcus granulosus and E. multilocularis: In vitro culture of the strobilar stages from protoscoleces. Angew. Parasit. 20 (1979) 137-147 13. Smyth, ]. D. and Z. Davies: In vitro culture of strobilar stages in Echinococcus granulosus (sheep strain): A review of basic poblems and results. Int. J. Parasitol. 4 (1974) 631-44 14. Smyth, J. D. and Z. Davies: In vitro suppression of segmentation in Echinococcus multilocularis with morphological transformation of protoscoleces into mono zoic adults. Parasitology 71 (1975) 125-135 15. Towbin, H., T. Staehlin, and]. Gordon: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Nat. Acad. Sci. USA 76 (1979) 4350-4354 16. Varela-Diaz, V. M.,]. A. Guisantes, M.]. Ricardes, L. A. Yarzabal, and E. A. Coltorti: Evaluation of whole and purified hydatid fluid antigens in the diagnosis of human hydatidosis by the immunoelectrophoresis test. Am. J. Trop. Med. Hyg. 24 (1975) 298-303 Dr. H. Auer, Department of Med. Parasitology, Institute of Hygiene, Kinderspitalgasse 15, A-1094 Vienna, Austria.