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Echinostoma caproni in mice: Shedding of antigens from the surface of an intestinal trematode
EClWVOSTOMA CAPIiONI IN MICE: SHEDDING OF ANTIGENS FROM THE SURFACE OF AN INTESTINAL TREMATODE K. ANDRESEN,* PAUL E. SIMONSEN,*~ B. JYDING ANDERSEN$ and A. BIRCH-A~DERSE~§ *Danish Bilharziasis Laboratory, Jaegersborg Alle 1 D, 2920 Charlottenhmd, Denmark *Department of Toxoplasmosis and §Uepartment of Biophysics, Statens Serum Institut, Amager Boulevard 80,230O Copenhagen S, Denmark (Received 21 June 1988; accepted 15 September 1988) K., SIMONSEN P. E., ANDERSEN B. J. and BIRCH-ANDERSEN A. 1989. Echinosf~m~ caproni in mice: shedding of antigens from the surface of an intestinal trematode. InternationaiJournalfor Pa~usitolo~ 19: 11 l-1 18. The surface antigens, which induce a serum antibody response during infection of mice with the ~testin~ trematode ~c~~~os~omu cuproni, were examined. It was demonstrated that antigens are shed from the surface of juvenile and 4-week old adult E. caproni during in vitro culture. SDSPAGE and Western blot analysis of in vitro shed and detergent solubilized surface antigens indicated that AbSfI3Cf-ANDRESEN
the four major antigens released from the surface of adult parasites had molecular masses of approximately 26,000,66,000,75,000 and 88,000. A modified ELISA technique showed the in vitro turn-over rate of the surface antigens to be very high, with a half-life of 8-15 min in both juvenile and adult E. caproni trematodes. Transmission electron microscopy of the surface of adult parasites revealed a highly active secreting tegument which was densely packed with membrane-bound vesicles, reflecting the high rate of shedding of the surface antigens. An attempt to immunize mice with detergent solubilized adult surface antigens failed to induce resistance to infection with metacerc~iae of E. caproni. INDEX KEY WORDS: Ec~~inosto~a cupruni; intestinal trematode; mice; in vitro culture; surface antigens; antigen shedding; surface turn-over; surface ultrastructure; immunization. INTRODUCTION
Echinostoma caproni is an intestinal trematode with no tissue phase in the final host. Mice are highly susceptible ta E. caproni infections, and the dynamics of this host/parasite combination have been examined in detail in our laboratory. Low ievei primary infections with less than 10 worms are expelled within 4-8 weeks, whereas primary infections with 15 or more worms are not expelled (Christensen, Nydal, Frandsen & Nansen, 1981; Odaibo, Christensen & Ukoli, 1988). A high level of resistance to reinfection develops approximately 2 weeks after a primary infection of any size (Sirag, Christensen, Frandsen, Monrad & Nansen, 1980; Christensen, Fagbemi & Nansen, 1984; Christensen, Knudsen & Andreassen, 1986), and several observations indicate that the resistance is of immunological nature (Christensen et al ., 1986; Simonsen & Andersen, 1986). Recent studies on the mechanisms of resistance in trematode infections, such as schistosomiasis (see reviews by McLaren, 1980; Butterworth, Taylor, Veith, Vadas, Dessein, Sturrock & Wells, 1982; Simpson & Smithers, 1985) and fascioliasis (see reviews by Reddington, Leid & Wescott, 1984; Hughes, 1983, have paid much attention to the hosts immune response against the parasite surface. These studies tTo whom correspondence
should be addressed.
have contributed significantly to the understanding of the mechanisms of host defence as well as on the parasites adaptations to avoid these mechanisms. Since both Schistosoma and Fasciolu undergo tissuemigration in the final host, however, knowledge obtained from studies on these trematodes may not be valid for purely intestinal trematodes. Infections with E. caproni in mice induce a serum antibody response against the surface of the parasites (Simonsen & Andersen, 1986). However, the surfacebound antibodies are rapidly lost from the live parasite surface during culturing in vitro, and it has been suggested that this might be due to shedding of antigens from the surface of the parasites (Simonsen & Andersen, 1986). The purpose of the following investigations was to demonstrate that surface antigens are shed from E. cuproni during in vitro culture, and to characterize the surface antigens according to molecular weight and turn-over rate. Furthermore, the ultrastructure of the surface of E. caproni was examined, and an attempt was made to immunize mice with E. cuproni surface antigens. LATERALS
AND ME~ODS
Mice and parasites. Inbred female BALE/c mice from Statens Serum Institut, Copenhagen, were used throughout this study. The mice were more than 6 weeks old and had an average weight of 17 g at the start of the experiments. The Echinos~oma population used in this study was originally 111
K. ANDRES~N, P. E. SIMONSYCN, B. J. ANDICRSENand A. BIRCH-ANDERSEN
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isolated in Egypt, and is identical to the population named E. revofutum in previous work on echinostomes from the Danish Bilharziasis Laboratory (e.g. Christensen et nl., 198 1,1984, 1986; Simonsen & Andersen 1986; Sirag et nl., 1980). However, a recent taxonomic revision of the genus Echinostoma by Kanev (unpublished Doctoral Thesis, University of Sofia, Bulgaria, 1985) shows that the correct name of this population is E. caproni. The parasites were maintained in the laboratory and infections of mice carried out as previously described (Simonsen & Andersen, 1986). Juvenile parasites were prepared by in vi&u excystation of metacercariae (Simonsen & Andersen, 1986). Serum. Antiserum was obtained from mice 28 days after infection with 1.5metacercariae. Sera from uninfected mice served as a control. Blood was collected by cardiac puncture under deep ether anaesthesia. Sera from mice in the same group were pooled and frozen at -7O’C until further use. In the first series of experiments, sera were precipitated with (NH&SO, before the immunogIobulin fraction was used. However, later all experiments were repeated using unfractionated sera, and the same results were obtained. Antigens. Four main antigen fractions were used in this study: excretory/secretory antigens obtained from juvenile (fraction 1) and adult (fraction 2) parasites cultured in vifro, and solubilized surface antigens obtained from juvenile (fraction 3) and adult (fraction 4) parasites treated with detergent in vitro, Adult worms were obtained from mice infected 28 days previously with 15 metacercariae. RPM1 1640 supplemented with 25 rnM HEPES, 22 mM NaHCO, and 50 ,~cgml-’ gentamy~ine (Garamy~in, Schering Corp., USA.) was used as the culture medium. The parasites were washed thoroughly in medium before in vitro culture. For isolation of excretory/secretory antigens, parasites were cultured in medium in concentrations of 5000 juveniles ml-’ or 10 adults ml-’ for 24 h at 37°C. After the incubation period, the medium was collected and frozen at -70°C (fraction 1 and 2, respectivelv). The parasites used for isola&on of solubilized ‘surface ‘antigens were first washed thoroughly in phosphate buffered saline, pH 7.4 (PBS) before thev were frozen at -20°C. After thawing, the parasites (10,000 juveniles mlIi or 20 adults ml-r) were placed in PBS containine 1% (v/v) Triton X-100 (Sigma, U.S.A.) and 100 kallikreic ina&vator units (k.i.u.) -ml-] apronnine (Trasylol, Bayer, West Germany) and incubated for 3 h on ice. The culture fluids were then centrifuged at 40,000 g for 30 min, and the supernatants frozen at -7O’C for later use (fractions 3 and 4). Precipitation in agarose gel. Ouchterlony immunodiffusion and crossed immunoelectrophoresis were carried out according to Jepsen & Axelsen (1980). SDS-PAGE and Western blot abuse. Sodium Dodecyl Sulphate-Polyacryl~ide Get Eiectrophorsis (SDS-PAGE) of the antigen fractions was carried out on a discontinuous gel-system (Laemmli, 1970) consisting of a lo-20% separation gel overlayered by a 5% stacking gel. Antigen samples (fractions 1-4) and molecular weight markers (same as used by Jakobsen, Theander, Jensen, Molbak & Jepsen, 1987) were boiled for 3 min in l/3 volume sample buffer, before being applied onto the gel. Electrophoresis was carried out at 7.5 mA overnight. The SDS-PAGE separated proteins were then electro~horetic~Iy transferred onto nitrocellulose paper ~itro~ellulose membrane, Amos, Denmark) usine a Semi Drv Multi Gel Electroblotter (Ancos, Denmark) according to Kyhse-Andersen (1984)‘The transfer time was 75 min at 0.5 mA cm->. After blocking and washing, the paper blots were incubated for 60 mitt in mouse antiserum (diluted 1: 45 in washing buffer). The blots were ”
then washed and incubated in peroxid~e-conjugated rabbit anti-mouse immunoglobulins (Dakopatts A/S: Denmark) diluted 1 :2000 in washing buffer. After further washing, the blots were stained in substrate solution (same as used for the ELISA procedure below) for 15 min. Blots with molecular markers were stained with colloidal gold. All experiments were carried out in duplicate and with both antiserum and control serum. Experiments were repeated with various amounts of antigen, and best results were obtained when 50100 yl of the boiled antigen solution was applied to the gel. Turn-over rate of surface antigens. A modified ELISAprocedure was adopted for measuring the turn-over rate of antigens located on the surface of juvenile (in vitro excysted) and adult (recovered 28 days after infection of mice with 25 metacercariae) parasites. The same culture medium as described for the antigen isolate above was used. Groups of five adults were incubated in a solution of 950 p I medium, 40 ~1 mouse antiserum and 10 fi I peroxidase-conjugated rabbit anti-mouse immunoglobuhns (Dakopatts A/S. Denmark) at 37°C for 20 min. Similarly, groups of 500 juveniles were incubated in haIf the volume (500~1) of the same solution. After incubation, the parasites were washed twice in 10 ml 37°C PBS and transferred to new tubes. The supernatant was removed and 1000 b I and 500 p 1 medium were added to adults and juveniles, respectively. At 5 min intervals thereafter, 3 X 20~1 samples were taken from each tube during the next 1 h. The volume of the medium containing the parasites was kept constant by addition of fresh medium after each three samples were taken. The 20,ul samples were transferred to flatbottomed microtest plates (Nunc, Denmark). and the relative amount of parasite antigen in the samples was estimated indirectly for each sample, as the amount of peroxidase-conjugated antibody oresent in the solution. To each well 200 11 of of substrate solution (41 mg o-phenylenediamine + 25 ~1 30% H,O, dissolved in 100 ml citrate ohosnhate buffer nH 5.0) was added, and the plates were iniubaied in darkness at 3O’C for 45 min. The reaction was stopped by addition of 50 ~1 of 3 MH$O, to each well, and the absorbance was measured at 490 nm. Control experiments were carried out similarly with control sera. Trwwnission electron microscopy. The surface of 28-day old adult E. caproni was studied by transmission electron microscopy. After recovery from the mouse intestines, the aarasites were washed five times in PBS, cut into three pieces (front, middle and rear part) and the pieces from each region fixed in 3% glutaraldehyde in cacodylate buffer pH 7.2 at 4°C overnight. Afterwards they were post-fixed in 1% osmium tetroxide in cacodylate buffer pH 7.2 for 1 h. followed by treatment for an additional hour with 2% uranyl acetate in Verona1 buffer pH 7.3. After dehydration in a series of ethanol, and treatment with propylene oxide, the tissue was embedded in Vestopal-W (Martin Jaeger, Switzerland) according to the routine procedures of the EM laboratory. Ultrathin sections were cut on an LKB Ultrotome III microtome and stained with magnesium uranyl acetate and lead citrate. Electron microscopy was carried out on a Philips E.M. 300 electron microscope operated at 60 kV. Immuri~zatjon of mice. Mice were immunized with solubilized adult worm antigen (fraction 4) four times with l-week intervals. The antigen solution was diluted with PBS containing 1% T&on X-100 to a protein content of 50 mg per injection. The first immunization (250 ~1, subcutaneous) contained a 1: 1 mixture of antigen solution and Freund’s complete adjuvant. For the second (subcutaneous), third and fourth (both intraperitoneally)
E. caproni surface antigens
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immunizations, only 100 ,ul antigen solution was injected. A control group received similar treatment but without antigen, whereas another control group did not receive any treatment at all. Twenty-eight days after the first immunization, all mice were infected orally with 25 metacercariae of E. caproni, and 28 days posf infection all mice were killed and the number of parasites in their intestines counted. RESULTS
Shedding of surface antigensfrom E. caproni Two types of E. caproni antigen fractions were isolated and analysed: (A) in vitro excretory/ secretory products released into the medium by live juvenile (fraction 1) and adult (fraction 2) worms, expected to contain a mixture of antigens from the surface, intestine and other structures; (B) detergent solubilized products from killed juvenile (fraction 3) and adult (fraction 4) parasites, expected to contain antigens from the surface only. Antigens isolated in both types of antigen fractions would therefore be expected to represent antigens released from the surface of live worms during culturing in vitro. All attempts to identify antigens in the four fractions by using Ouchterlony immunodiffusion and crossed immunoelectrophoresis with various concentrations of mouse antiserum and antigen failed, in that no visible precipitates appeared in the gels. In contrast, using SDS-PAGE and Western blot analysis, antigens were identified in all fractions (an example is shown in Fig. 1). Several antigens were identified in the adult fractions, and four of these showed up very clearly both with the adult excretory/ secretory fraction (fraction 2) and with the adult detergent solubilized fraction (fraction 4) indicating that these four antigens represent the major antigens shed from the surface of adult worms during in vitro culture. The molecular masses of these four antigens were estimated to be approximately 26,000, 66,000, 75,000 and 88,000. Additional antigens were frequently identified in both of the adult fractions, representing antigens with molecular masses of approximately 32,000, 38,000, 41,000, 43,000 and 155,000. No antigens were identified exclusively in the excretory/secretory or the detergent solubilized fractions. With juvenile antigen fractions, only very faint antigen-antibody reactions were observed, and none of these were seen in both the excretory/secretory fraction (fraction 1) and in the detergent solubilized fraction (fraction 3). Of the major adult worm surface antigens, the 26,000 mass antigen was identified in the juvenile antigen fraction 3, whereas the 75,000 antigen was identified in the juvenile antigen fraction 1. None of the above antigens were identified with the use of control sera. Turn-over rate of the surface antigens The rate of antigen shedding from the surface of juvenile and 28-day old adult E. caproni was measured indirectly, as the rate by which surfacebound antibodies were released from the parasites
FIG. 1. Western blot analysis of E. caproni surface antigens. The molecular masses of the identified major surface antigens are indicated on the right in thousands. (a) Juvenile detergent solubilized antigens (fraction 3); (b) adult detergent solubilized antigens (fraction 4); (c) adult excretory/secretory antigens (fraction 2).
into the surrounding medium. Live parasites were incubated in medium containing mouse antiserum and peroxidase conjugated anti-mouse immunoglobulins. They were then washed and immediately transferred to fresh medium for culture. The relative amount of peroxidase released into the medium was measured by our modified ELISA, and used as an estimate of the amount of shed antigens. Three experiments were carried out in triplicate, using antisera and control sera, and similar results were obtained each time. Results from one of the experiments are presented in Fig. 2. For both adult and juvenile parasites, a maximum concentration of shed peroxidase-conjugated antibody was reached approximately 20-25 min after the parasites had been transferred to fresh medium, indicating that no more surface-bound antibody was released from the parasites after this period. The time for release of half of the surface-bound antibody (the half-life) varied from 8 to 15 min in the three experiments, and was always shorter for adults than for juveniles in the same experiments, although this difference was not statistically significant. With the control sera, no antibody was released from the surface of juvenile parasites. However, a significant amount of antibody was
114
K.
ELI:
absorbance
ANDRESEN,
P. E.
SIMONSEN,
B. J. ANDERSEN
values (A49o x 103)
1
a. 60
0
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ELISA absorbance
100-
20
40
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values (A490 x 10
50
60
so-
60-
10
20
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BIRCH-ANDERSEN
body in the culture medium was reached. Juvenile parasites were labelled with anti-surface and peroxidase-conjugated antibodies, washed and cultured in fresh medium as described above. After 0 and 45 min, worms were transferred to new tubes with fresh medium, substrate was added directly to the parasites, and after 30 min samples of the supernatant were examined for colour-reaction on the ELISAreader. The absorbance values for juveniles cultured for 0 min were positive (mean of 108 X 10m3)whereas juveniles cultured for 45 min had absorbance values of zero, indicating that all surface-bound antibody had been released from the surface of the juveniles during the 4.5 min of culture. Transmission electron microscopy
)
b.
0
and A.
40
50
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FIG. 2. Relative amount of released surface-bound antibody from E. caproni, as measured by the described modified EL&A. The graphs indicate mean f S.D. of experiments carried out in triplicate. (a) Adult worms incubated in immune (m) and control (0) serum. (b) Juvenile worms incubated in immune (m) and control (0) serum.
released from adults in control cultures (Fig. 2), probably originating from the host-mice (and not from the control sera), since the serum of mice with 2%day old infections are known to contain antibodies against the E. caproni surface. An experiment was set up to examine if all the surface-bound antibody had been released from the juvenile parasites when the maximum level of anti-
To examine if the high rate of antigen shedding was reflected in the parasite ultrastructure, the surface of adult E. caprooni was studied by transmission electron microscopy. A typical trematode tegument was observed (Fig. 3a), which was highly folded and covered with microvilli on the outside. The cytoplasm of the tegument contained spines and numerous small vesicles. Higher magnification (Figs. 3b, c and d) showed the tegument to be covered distally by a trilaminate plasma membrane with a thin layer of glycocalyx on the exterior. Towards the interior of the parasite, the tegumental cytoplasm was lined by a plasma membrane resting on a basal lamina. Cytoplasmic tubes traversed the basal lamina and connected the distal syncytial tegument to the proximal cell bodies. The tegument contained large numbers of membrane-bound vesicles, especially lined up near the outer surface of the parasite, thus indicating a very active secreting surface. At least two morphological types of membr~e-bound vesicles were identi~ed in the tegument: elongate vesicles of 150-200 X 3050 nm in size, and circular vesicles with a diameter of 150-200 nm. The interior of both types of vesicles usually contained a core with strands of intermediate electron dense material, and a periphery with a more electron dense substance. Elongate vesicles were most frequently observed in the distal part of the tegument, whereas the frequency of circular vesicles was highest further interior. Membrane-bound vesicles of various sizes were furthermore often observed exterior to the surface of the parasite (Figs. 3b, c and d). These vesicles appeared too small to be merely sections through the microvilli without the stalks appearing in the plane of section, and they may represent material released from the parasite surface.
FIG. 3. Transmission electron microscopy of the surface of 2%day old adult E. caproni. (a) Overview of the tegument showing tegumental cytoplasm with organelles (TC) and spine (S), basal Iamina (BL) and microvilli (MV). Scale bar = 5 pm. (b) Microvilli on the distal part of the tegument showing plasma membrane (PM), glycocalyx (GX), elongate vesicles (EV) and extracellular vesicles (XV). Scale bar = 200 MI. (c) Microvilli on distal part of the tegument showing plasma membrane (PM), glycocalyx (GX), elongate vesicles (EV), circular vesicles (CV) and extracellular vesicles (XV). Scale bar = 200 nm. (d) Distal part of the tegument. MicrovilIi densely packed with elongate (EV) and circular (CV) vesicles. Scale bar = 200 nm.
E. caproni surface antigens
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K.
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ANDRESEN,
P. E.
SIMONSEN, B.
J.
Parasites recovered 25
4. Recovery of E. caproni from the intestines of mice immunized with detergent solubilized adult surface antigens (fraction 4) and subsequently infected orally with 25 metacercariae. Histo)?;ram indicates mean f S.D. (a) Control group, without keund’s complete adjuvant ‘@CA). (b) FIG.
Control group, with FCA. (c) Experimental group, immunized with antigen fraction 4 + FCA.
IrnrnLi~i~~t~o~with q&ace antigens An attempt was made to immunize mice against reinfection with metacercariae of E. caproni by using adult detergent solubilized surface antigen (antigen fraction 4). Ten mice were immunized with antigen in adjuvant, five control mice received adjuvant only, whereas five control mice did not receive any treatment at all. Four weeks after the first i~unization, all mice were infected orally with 25 metacercariae of E. eapruni. The results are shown in Fig. 4. No resistance to a subsequent infection with E. caproni metacercariae was observed in the immunized mice. DISCUSSION Infections with E. cuproni in mice induce a serum antibody response to the surface of the parasites (Simonsen & Andersen, 1986). However, the antibodies are rapidly lost from the live parasite surface during culturing in vitro, and it has been suggested that this could be due to shedding of surface antigens (Simonsen & Andersen, 1986). The present study demonstrated that antigens are indeed released from the adult E. caproni surface during in vitro culture. Furthermore, all excretory/ secretory antigens recovered from adults had their origin on the worm surface, since they were ail detected in the detergent solubilized surface-antigen fractions as well. If antigens are also released from the intestine or other structures of the parasites, they must be similar to the surface antigens, or they are released in quantities too low to be detected with the employed methods. Four major surface antigens were observed
ANDERSEN
and A.
BIRCH-ANDERSEN
in adult worms, i.e. antigens that consistently showed dense and clear bands in all electrophoretic experiments performed. Additional weaker bands were frequently observed, and these we believe represent minor adult surface antigens. Juvenile antigen fractions revealed only two very weak bands in the electrophoretic experiments, probably due to low antigen concentrations. One antigen was observed in the detergent solubilized fraction only, indicating that this antigen originated on the juvenile surface. Furthermore, this antigen was similar in molecular mass to one of the major adult antigens. Another band observed with the juvenile in vitro antigen fraction corresponded to another of the major adult surface antigens, and therefore probably also originated on the juvenile surface. Two surface antigens were therefore demonstrated on juvenile El. cuproni, both similar in electrophoretic pattern to adult major surface antigens, and at least one of these was released from the juveniles during in vitro culture. The shedding of antigens from the surface of E. caproni may explain the previously described loss of antibodies and attacking cells from the surface of these parasites during in vitro culture (Simonsen B Andersen, 1986). Furthermore, the ability of adult E. cuproni from primary infections to survive for long periods in i~une mice (Sirag et al., 1980; Christensen et al., 1984) may be due to antigen shedding, thus rendering the worms inaccessible to the hosts immune system. The phenomenon of surface antigen shedding during in vitro culture has also been reported for the trematodes F. hepatica (see Hanna, 1980; Lammas Cyr. Duffus, 1983) and S. mansoni (see Kusel, Mackenzie & McLaren, 1975; Wilson & Barnes, 1977; Samuelson, Sher & Caulfield, 1980; Ruppel & McLaren, 1986), and there is accumula~ng evidence that antigen shedding in these parasites is occurring in vivo as well (Burden, Bland, Hammet & Hughes, 1983; Pearce, Basch & Sher, 1986; Ruppel & McLaren, 1986), as an adaptation to withstand the hosts immune attack. The in vitro turn-over rate of E. caproni surface antigens was found by our modified ELISA to be very high, with a half-life of 8-15 min for both adult and juvenile parasites. Furtherm~)re, a control study on juvenile parasites indicated that all surface antigens were released within 45 min. Previous observations on the loss of surface-bound antibodies from juvenile E. caproni, using immunofluorescence microscopy (IFAT), had indicated a slower rate of release, leaving 4-5 h before all antibody was lost from the surface (Simonsen & Andersen, 1986). This discrepancy may be due to different culturing conditions as well as to different sensitivities of the techniques emptoyed in the two studies. The ELISA studies supported the observations from previous IFAT studies (Simonsen & Andersen, 1986) that the surface of adult E. cuproni is covered with antibody when the worms are removed from the
117
E. caproni surface antigens
mouse intestine 4 weeks post primary infection. Since adult E. cuproni from primary infections are not expelled, or otherwise seem to be affected by the hosts immune system at 4 weeks after infection, the antibodies against the surface do not seem to have an immediate effect on the adult worms in viva. Continued release of antigens from the E. cuproni surface in vivo will cause a significant antigenic stimulus to the host. However, immunization of mice with detergent solubilized surface antigens from adult worms in the present study did not lead to any reduction in the worm establishment in a subsequent oral infection with metacercariae of E. cuproni. This failure may be related to the immunization procedure and to the nature of the antigen. Since the parasites are purely intestinal, the antigens may need local intestinal presentation (Befus & Bienenstock, 1982) instead of subcutaneous or intraperitoneal injection. Furthermore, antigens produced from juvenile parasites may be more potent stimulators of resistance, since it is known from previous experiments that resistance is directed against the juvenile parasites (Christensen et al., 1986). Similar immunization of rodents with excretory/secretory products of Fusciola heputicu (see Lehner & Sewell, 1979; Rajasekariah, Mitchell, Chapman & Montague, 1979; Burden, Harness & Hammet, 1982) have generally yielded better results when antigens were derived from juvenile or young flukes than from adults. In the present study it was not possible to produce sufficient amounts of juvenile antigen for this purpose. Resistance-related factors such as reduction in growth or reproductive capacity or reduced pathology due to infection were not examined in the present investigation, but may well have been affected by the immunization. The EM studies on the surface of adult E. caproni revealed a highly active tegument, and thereby reflected the high rate of antigen shedding from the parasite surface. In gross structure, the tegument was similar to what has been described from F. hepaticu (see Threadgold, 1963, 1967, 1984), with a distal syncytial cytoplasm containing organelles and spines, and limited outwards by a trilaminate plasma membrane covered by a thin glycocalyx. The tegument was densely packed with membrane bound vesicles of at least two morphological types, which were lined up along the outer membrane, the elongate vesicles usually perpendicular to the membrane. It is tempting to suggest that these vesicles contain antigenic material which is released onto the parasite surface, thereby maintaining the continued antigen shedding. Small membrane-bound vesicles were furthermore observed outside the parasite. These vesicles may contain material released from the parasite surface. More detailed ultrastructural studies on the dynamics of the E. cuproni tegument are in progress. Acknowledgements-The following are valuable assistance and discussions during
thanked for the course of
these investigations: Dr Palle H. Jakobsen and his staff in the Department of Treponematosis, Statens Serum Institut, Mrs Helene Ravn, Department of Biophysics, Statens Serum Institut, Dr Jerrn Andreassen, Institute of Population Biology, University of Copenhagen, and Dr Niels 0. Christensen, Danish Bilharziasis Laboratory. The investigations were supported by grants from the Danish Natural Science Research Council.
REFERENCES BEFUS D. & BIENENSTOCK J. 1982. Factors involved in symbiosis and host resistance at the mucosal-parasite interface. Progress in Allerav 31: 76-177. BURDEN D. J., I&NESS E. ~‘HAMMET N. C. 1982. Fasciola hepafica: attempts to immunize rats and mice with metabolic and somatic antigens derived from juvenile flukes. Veterinay Parasitology 9: 26 l-266. BURDEN D. J., BLAND A. P., HAMMET N. C. & HUGHES D. L. 1983. Fasciola heuafica: mieration of newlv excysted juveniles in resistant rats. Experimental Parasitology 56: 277-288. BUTT~RWORTHA. E., TAYLOR D. W., VEITH M. C., VADAS M. A., DESSEIN A., STURROCKR. F. & WELLS E. 1982. Studies on the mechanisms of immunity in human schistosomiasis. Immunological Reviews 61: 5-39. CHRISTENSEN N. 0., NYDAL R., FKANDSEN F. & NANSEN P. I98 I. Homologous immunotolerance and decreased resistance to Schistosoma mansoni in Echinostoma revolutum-infected mice. Journal of Parasirology 61: 164-166. CHRISTENSEN N. 0.. FAGBEMI B. 0. & NANSEN P. 1984. TTpanosoma brucei-induced blockage of expulsion of Echinosloma revolutum and of homologous E. revoluturn resistance in mice. Journal ofParusitology 70: 55% 561. CHRISTENSEN N. O., KNUDSEN J. & ANDREASSEN J. 1986. Echinostoma revolulum: resistance to secondary and superimposed infections in mice. Experimental Parasitology61:311-318. HANNA R. E. B. 1980. Fasciola hepatica: glycocalyx replacement in the juvenile as a possible mechanism for protection against host immunity. . Experimental Para. &ology 50: io3- i 14. HUGHES D. L. 1985. Trematodes, excluding schistosomes with special emphasis on Fasciola. In: Parasite Antigens in Prote&ion, LIia&osis and Escape (Edited by PARKHOUSE R. M. E.). Currem Topics in Microbiology and Immunology 120: 24 I-260. JAKOBSEN P. H., THEANDER T. G., JENSEN J. B., MBLBAK K. & J~PSEN S. 1987. Soluble Plasmodium falciparum antigens contain carbohydrate moieties important for immune reactivity. Journal of Clinical Microbiolog?, 25: 2075-2079. JEPSEN S. & AXELSEN N. H. 1980. Antigens and antibodies in Plasmodium falciparum malaria studied by the immunoelectrophoretic method. Acta Pathologica, Microbiologica et Immunologica Scandinavica, Section C 88: 263-270. KUSEL J. R., MACKENZIE P. E. &MCLAREN D. J. 197.5.The release of membrane antigens into culture by adult Schistosoma mansoni. Parasitology 11: 247-259. KYHSE-ANDERSEN J. 1984. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer I
ll8
K.
ANDRESEN,
of proteins from po~yac~~~de
P. E.
SZMONSEN,B.
J.
to &trocefIulose. Joldrnai
~f~~~c~emical and 3jop~ys~cul Methods 10: 203-209. LAEMMLI U. K.
1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: 680-685. LAMMASD. A. & DUFFUSW. P. H. 1983. The shedding of the outer glycocatyx of juvenile Fasciola hepatica. Veterinary Parasitology 12: 165-l 78.
LEHN~R R. P. & SEWELL M. M.H. 1979. Attempted immunisation of laboratory animals with metabolic antigens of Fusciofn ~epat~~a. Veterinary Science Communi~a~~~~ 2: 337-340.
MCLAREND. J. 1980. Schistosoma mansoni: The Parasite Surface in Relation to Host Immunity. Research Studies Press, John Wiley, New York. ODAIBOA. B., CHRISTENSEN N. 0. & UKCILIF. M. A. 1988. Establishment, survival and fecundity in Echinosfoma caproni infections in NMRI mice. Proceedinns of the H~lm~~thol~~ical Society of Wash~~~t~~~ 55: 26<-289. PEMCE E. J.. BASCH P. F. & SEER A. 1986. Evidence that the r&u&d surface antigenicity of developing fch&osoma mansoni schistosomula is due to antigen shedding rather than host molecule acquisition. Parasite Immunology 8: 79-94.
RAJASEKARIA~ G. R., MITCHELLG. F., CHAPMANC. B. & MONTAGUEP. E. 1979. Fasciola hepatica: attempts to induce orotection against infection in rats and mice by injection of excreto$secretory products of immature worms. Parasitolo~ 79: 393-400. REDDINGTON J. J., Lim R. W. & WEXOTT R. B. 1984. A review of the antigens of Fasciola hepatica. ~ter~~a~ Par~itolo~ 14: 209-229. RUPPEL A. & MCLAREN D. J. 1986. Schktosoma mansoni:
ANDERSEN
and A. BIRCH-ANDERSEN
surface membrane stability tn vitro and in v&o. Experimen~ul Parusitology $2: 223-236. SAMUELSON J. C., SHERA. & CMJLPIELD.i. P. 1980. Newly transformed schistosomula spontaneously lose surface antigens and C3 acceptor sites during culture. Journal of Immunology 124: 20552057. SIMONSEN P. E. & ANDERSENB. J. 1986. Echinostoma rev&turn in mice; dynamics of the antibody attack to the surface of an intestinal trematode. ~~e~~~r~o~a~Journal .~orPff~~ol~~ 16: 475-482, SIMPS~~~A. 3. G. C SYIJTHERS S. R. 1985. Schistosomes: surface, egg and circulating antigens. In: Parasite Antigens in Protection, Diagnosis and Escape (Edited by PARK~ONSB R. M. E.). Current Topics in Microbiology and Immunology 120: 205-239. &RAG S. EL,?IHRISTENSEN N. Is)., FRANDSENF., MONRAD J. & NANSENP. 1980. Homologous and heterologous resistance in Echinosroma revo&um infections in mice, Parasitolo~
80: 479-486.
THREADGOLD LT. 1963. The tegument and associated structures of Firs&la hepatica. quarterly Jourzal of microscopical Science 104: SQ5-5 12. THPEALYGOLDL.
T. 1967. Electron microscope studies of Further observations on the tegument and associated structures. Parasitology 57: Fasciola
hepatica-III.
633-637.
THREADGOLDL. T.
1984. Parasitic platyhelminths. In: Vol. 1 (Ehiteh by BEREITERHAHN J.. M~TOLTS~A. G. & RICHARDS K. S.f., I. DV. 13219 1. Springer, Berlin. WILSON R. A. & BAENE~ P. E. 1977. The formation and turnover of the membr~acalyx on the tegument of Biology of the fntegument.
Schistosoma mansoni. Parasitology 74: 6 i-7 1.
the four major antigens released from the surface of adult parasites had molecular masses of approximately 26,000,66,000,75,000 and 88,000. A modified ELISA technique showed the in vitro turn-over rate of the surface antigens to be very high, with a half-life of 8-15 min in both juvenile and adult E. caproni trematodes. Transmission electron microscopy of the surface of adult parasites revealed a highly active secreting tegument which was densely packed with membrane-bound vesicles, reflecting the high rate of shedding of the surface antigens. An attempt to immunize mice with detergent solubilized adult surface antigens failed to induce resistance to infection with metacerc~iae of E. caproni. INDEX KEY WORDS: Ec~~inosto~a cupruni; intestinal trematode; mice; in vitro culture; surface antigens; antigen shedding; surface turn-over; surface ultrastructure; immunization. INTRODUCTION
Echinostoma caproni is an intestinal trematode with no tissue phase in the final host. Mice are highly susceptible ta E. caproni infections, and the dynamics of this host/parasite combination have been examined in detail in our laboratory. Low ievei primary infections with less than 10 worms are expelled within 4-8 weeks, whereas primary infections with 15 or more worms are not expelled (Christensen, Nydal, Frandsen & Nansen, 1981; Odaibo, Christensen & Ukoli, 1988). A high level of resistance to reinfection develops approximately 2 weeks after a primary infection of any size (Sirag, Christensen, Frandsen, Monrad & Nansen, 1980; Christensen, Fagbemi & Nansen, 1984; Christensen, Knudsen & Andreassen, 1986), and several observations indicate that the resistance is of immunological nature (Christensen et al ., 1986; Simonsen & Andersen, 1986). Recent studies on the mechanisms of resistance in trematode infections, such as schistosomiasis (see reviews by McLaren, 1980; Butterworth, Taylor, Veith, Vadas, Dessein, Sturrock & Wells, 1982; Simpson & Smithers, 1985) and fascioliasis (see reviews by Reddington, Leid & Wescott, 1984; Hughes, 1983, have paid much attention to the hosts immune response against the parasite surface. These studies tTo whom correspondence
should be addressed.
have contributed significantly to the understanding of the mechanisms of host defence as well as on the parasites adaptations to avoid these mechanisms. Since both Schistosoma and Fasciolu undergo tissuemigration in the final host, however, knowledge obtained from studies on these trematodes may not be valid for purely intestinal trematodes. Infections with E. caproni in mice induce a serum antibody response against the surface of the parasites (Simonsen & Andersen, 1986). However, the surfacebound antibodies are rapidly lost from the live parasite surface during culturing in vitro, and it has been suggested that this might be due to shedding of antigens from the surface of the parasites (Simonsen & Andersen, 1986). The purpose of the following investigations was to demonstrate that surface antigens are shed from E. cuproni during in vitro culture, and to characterize the surface antigens according to molecular weight and turn-over rate. Furthermore, the ultrastructure of the surface of E. caproni was examined, and an attempt was made to immunize mice with E. cuproni surface antigens. LATERALS
AND ME~ODS
Mice and parasites. Inbred female BALE/c mice from Statens Serum Institut, Copenhagen, were used throughout this study. The mice were more than 6 weeks old and had an average weight of 17 g at the start of the experiments. The Echinos~oma population used in this study was originally 111
K. ANDRES~N, P. E. SIMONSYCN, B. J. ANDICRSENand A. BIRCH-ANDERSEN
112
isolated in Egypt, and is identical to the population named E. revofutum in previous work on echinostomes from the Danish Bilharziasis Laboratory (e.g. Christensen et nl., 198 1,1984, 1986; Simonsen & Andersen 1986; Sirag et nl., 1980). However, a recent taxonomic revision of the genus Echinostoma by Kanev (unpublished Doctoral Thesis, University of Sofia, Bulgaria, 1985) shows that the correct name of this population is E. caproni. The parasites were maintained in the laboratory and infections of mice carried out as previously described (Simonsen & Andersen, 1986). Juvenile parasites were prepared by in vi&u excystation of metacercariae (Simonsen & Andersen, 1986). Serum. Antiserum was obtained from mice 28 days after infection with 1.5metacercariae. Sera from uninfected mice served as a control. Blood was collected by cardiac puncture under deep ether anaesthesia. Sera from mice in the same group were pooled and frozen at -7O’C until further use. In the first series of experiments, sera were precipitated with (NH&SO, before the immunogIobulin fraction was used. However, later all experiments were repeated using unfractionated sera, and the same results were obtained. Antigens. Four main antigen fractions were used in this study: excretory/secretory antigens obtained from juvenile (fraction 1) and adult (fraction 2) parasites cultured in vifro, and solubilized surface antigens obtained from juvenile (fraction 3) and adult (fraction 4) parasites treated with detergent in vitro, Adult worms were obtained from mice infected 28 days previously with 15 metacercariae. RPM1 1640 supplemented with 25 rnM HEPES, 22 mM NaHCO, and 50 ,~cgml-’ gentamy~ine (Garamy~in, Schering Corp., USA.) was used as the culture medium. The parasites were washed thoroughly in medium before in vitro culture. For isolation of excretory/secretory antigens, parasites were cultured in medium in concentrations of 5000 juveniles ml-’ or 10 adults ml-’ for 24 h at 37°C. After the incubation period, the medium was collected and frozen at -70°C (fraction 1 and 2, respectivelv). The parasites used for isola&on of solubilized ‘surface ‘antigens were first washed thoroughly in phosphate buffered saline, pH 7.4 (PBS) before thev were frozen at -20°C. After thawing, the parasites (10,000 juveniles mlIi or 20 adults ml-r) were placed in PBS containine 1% (v/v) Triton X-100 (Sigma, U.S.A.) and 100 kallikreic ina&vator units (k.i.u.) -ml-] apronnine (Trasylol, Bayer, West Germany) and incubated for 3 h on ice. The culture fluids were then centrifuged at 40,000 g for 30 min, and the supernatants frozen at -7O’C for later use (fractions 3 and 4). Precipitation in agarose gel. Ouchterlony immunodiffusion and crossed immunoelectrophoresis were carried out according to Jepsen & Axelsen (1980). SDS-PAGE and Western blot abuse. Sodium Dodecyl Sulphate-Polyacryl~ide Get Eiectrophorsis (SDS-PAGE) of the antigen fractions was carried out on a discontinuous gel-system (Laemmli, 1970) consisting of a lo-20% separation gel overlayered by a 5% stacking gel. Antigen samples (fractions 1-4) and molecular weight markers (same as used by Jakobsen, Theander, Jensen, Molbak & Jepsen, 1987) were boiled for 3 min in l/3 volume sample buffer, before being applied onto the gel. Electrophoresis was carried out at 7.5 mA overnight. The SDS-PAGE separated proteins were then electro~horetic~Iy transferred onto nitrocellulose paper ~itro~ellulose membrane, Amos, Denmark) usine a Semi Drv Multi Gel Electroblotter (Ancos, Denmark) according to Kyhse-Andersen (1984)‘The transfer time was 75 min at 0.5 mA cm->. After blocking and washing, the paper blots were incubated for 60 mitt in mouse antiserum (diluted 1: 45 in washing buffer). The blots were ”
then washed and incubated in peroxid~e-conjugated rabbit anti-mouse immunoglobulins (Dakopatts A/S: Denmark) diluted 1 :2000 in washing buffer. After further washing, the blots were stained in substrate solution (same as used for the ELISA procedure below) for 15 min. Blots with molecular markers were stained with colloidal gold. All experiments were carried out in duplicate and with both antiserum and control serum. Experiments were repeated with various amounts of antigen, and best results were obtained when 50100 yl of the boiled antigen solution was applied to the gel. Turn-over rate of surface antigens. A modified ELISAprocedure was adopted for measuring the turn-over rate of antigens located on the surface of juvenile (in vitro excysted) and adult (recovered 28 days after infection of mice with 25 metacercariae) parasites. The same culture medium as described for the antigen isolate above was used. Groups of five adults were incubated in a solution of 950 p I medium, 40 ~1 mouse antiserum and 10 fi I peroxidase-conjugated rabbit anti-mouse immunoglobuhns (Dakopatts A/S. Denmark) at 37°C for 20 min. Similarly, groups of 500 juveniles were incubated in haIf the volume (500~1) of the same solution. After incubation, the parasites were washed twice in 10 ml 37°C PBS and transferred to new tubes. The supernatant was removed and 1000 b I and 500 p 1 medium were added to adults and juveniles, respectively. At 5 min intervals thereafter, 3 X 20~1 samples were taken from each tube during the next 1 h. The volume of the medium containing the parasites was kept constant by addition of fresh medium after each three samples were taken. The 20,ul samples were transferred to flatbottomed microtest plates (Nunc, Denmark). and the relative amount of parasite antigen in the samples was estimated indirectly for each sample, as the amount of peroxidase-conjugated antibody oresent in the solution. To each well 200 11 of of substrate solution (41 mg o-phenylenediamine + 25 ~1 30% H,O, dissolved in 100 ml citrate ohosnhate buffer nH 5.0) was added, and the plates were iniubaied in darkness at 3O’C for 45 min. The reaction was stopped by addition of 50 ~1 of 3 MH$O, to each well, and the absorbance was measured at 490 nm. Control experiments were carried out similarly with control sera. Trwwnission electron microscopy. The surface of 28-day old adult E. caproni was studied by transmission electron microscopy. After recovery from the mouse intestines, the aarasites were washed five times in PBS, cut into three pieces (front, middle and rear part) and the pieces from each region fixed in 3% glutaraldehyde in cacodylate buffer pH 7.2 at 4°C overnight. Afterwards they were post-fixed in 1% osmium tetroxide in cacodylate buffer pH 7.2 for 1 h. followed by treatment for an additional hour with 2% uranyl acetate in Verona1 buffer pH 7.3. After dehydration in a series of ethanol, and treatment with propylene oxide, the tissue was embedded in Vestopal-W (Martin Jaeger, Switzerland) according to the routine procedures of the EM laboratory. Ultrathin sections were cut on an LKB Ultrotome III microtome and stained with magnesium uranyl acetate and lead citrate. Electron microscopy was carried out on a Philips E.M. 300 electron microscope operated at 60 kV. Immuri~zatjon of mice. Mice were immunized with solubilized adult worm antigen (fraction 4) four times with l-week intervals. The antigen solution was diluted with PBS containing 1% T&on X-100 to a protein content of 50 mg per injection. The first immunization (250 ~1, subcutaneous) contained a 1: 1 mixture of antigen solution and Freund’s complete adjuvant. For the second (subcutaneous), third and fourth (both intraperitoneally)
E. caproni surface antigens
113
immunizations, only 100 ,ul antigen solution was injected. A control group received similar treatment but without antigen, whereas another control group did not receive any treatment at all. Twenty-eight days after the first immunization, all mice were infected orally with 25 metacercariae of E. caproni, and 28 days posf infection all mice were killed and the number of parasites in their intestines counted. RESULTS
Shedding of surface antigensfrom E. caproni Two types of E. caproni antigen fractions were isolated and analysed: (A) in vitro excretory/ secretory products released into the medium by live juvenile (fraction 1) and adult (fraction 2) worms, expected to contain a mixture of antigens from the surface, intestine and other structures; (B) detergent solubilized products from killed juvenile (fraction 3) and adult (fraction 4) parasites, expected to contain antigens from the surface only. Antigens isolated in both types of antigen fractions would therefore be expected to represent antigens released from the surface of live worms during culturing in vitro. All attempts to identify antigens in the four fractions by using Ouchterlony immunodiffusion and crossed immunoelectrophoresis with various concentrations of mouse antiserum and antigen failed, in that no visible precipitates appeared in the gels. In contrast, using SDS-PAGE and Western blot analysis, antigens were identified in all fractions (an example is shown in Fig. 1). Several antigens were identified in the adult fractions, and four of these showed up very clearly both with the adult excretory/ secretory fraction (fraction 2) and with the adult detergent solubilized fraction (fraction 4) indicating that these four antigens represent the major antigens shed from the surface of adult worms during in vitro culture. The molecular masses of these four antigens were estimated to be approximately 26,000, 66,000, 75,000 and 88,000. Additional antigens were frequently identified in both of the adult fractions, representing antigens with molecular masses of approximately 32,000, 38,000, 41,000, 43,000 and 155,000. No antigens were identified exclusively in the excretory/secretory or the detergent solubilized fractions. With juvenile antigen fractions, only very faint antigen-antibody reactions were observed, and none of these were seen in both the excretory/secretory fraction (fraction 1) and in the detergent solubilized fraction (fraction 3). Of the major adult worm surface antigens, the 26,000 mass antigen was identified in the juvenile antigen fraction 3, whereas the 75,000 antigen was identified in the juvenile antigen fraction 1. None of the above antigens were identified with the use of control sera. Turn-over rate of the surface antigens The rate of antigen shedding from the surface of juvenile and 28-day old adult E. caproni was measured indirectly, as the rate by which surfacebound antibodies were released from the parasites
FIG. 1. Western blot analysis of E. caproni surface antigens. The molecular masses of the identified major surface antigens are indicated on the right in thousands. (a) Juvenile detergent solubilized antigens (fraction 3); (b) adult detergent solubilized antigens (fraction 4); (c) adult excretory/secretory antigens (fraction 2).
into the surrounding medium. Live parasites were incubated in medium containing mouse antiserum and peroxidase conjugated anti-mouse immunoglobulins. They were then washed and immediately transferred to fresh medium for culture. The relative amount of peroxidase released into the medium was measured by our modified ELISA, and used as an estimate of the amount of shed antigens. Three experiments were carried out in triplicate, using antisera and control sera, and similar results were obtained each time. Results from one of the experiments are presented in Fig. 2. For both adult and juvenile parasites, a maximum concentration of shed peroxidase-conjugated antibody was reached approximately 20-25 min after the parasites had been transferred to fresh medium, indicating that no more surface-bound antibody was released from the parasites after this period. The time for release of half of the surface-bound antibody (the half-life) varied from 8 to 15 min in the three experiments, and was always shorter for adults than for juveniles in the same experiments, although this difference was not statistically significant. With the control sera, no antibody was released from the surface of juvenile parasites. However, a significant amount of antibody was
114
K.
ELI:
absorbance
ANDRESEN,
P. E.
SIMONSEN,
B. J. ANDERSEN
values (A49o x 103)
1
a. 60
0
10
ELISA absorbance
100-
20
40
30
values (A490 x 10
50
60
so-
60-
10
20
30
BIRCH-ANDERSEN
body in the culture medium was reached. Juvenile parasites were labelled with anti-surface and peroxidase-conjugated antibodies, washed and cultured in fresh medium as described above. After 0 and 45 min, worms were transferred to new tubes with fresh medium, substrate was added directly to the parasites, and after 30 min samples of the supernatant were examined for colour-reaction on the ELISAreader. The absorbance values for juveniles cultured for 0 min were positive (mean of 108 X 10m3)whereas juveniles cultured for 45 min had absorbance values of zero, indicating that all surface-bound antibody had been released from the surface of the juveniles during the 4.5 min of culture. Transmission electron microscopy
)
b.
0
and A.
40
50
60
FIG. 2. Relative amount of released surface-bound antibody from E. caproni, as measured by the described modified EL&A. The graphs indicate mean f S.D. of experiments carried out in triplicate. (a) Adult worms incubated in immune (m) and control (0) serum. (b) Juvenile worms incubated in immune (m) and control (0) serum.
released from adults in control cultures (Fig. 2), probably originating from the host-mice (and not from the control sera), since the serum of mice with 2%day old infections are known to contain antibodies against the E. caproni surface. An experiment was set up to examine if all the surface-bound antibody had been released from the juvenile parasites when the maximum level of anti-
To examine if the high rate of antigen shedding was reflected in the parasite ultrastructure, the surface of adult E. caprooni was studied by transmission electron microscopy. A typical trematode tegument was observed (Fig. 3a), which was highly folded and covered with microvilli on the outside. The cytoplasm of the tegument contained spines and numerous small vesicles. Higher magnification (Figs. 3b, c and d) showed the tegument to be covered distally by a trilaminate plasma membrane with a thin layer of glycocalyx on the exterior. Towards the interior of the parasite, the tegumental cytoplasm was lined by a plasma membrane resting on a basal lamina. Cytoplasmic tubes traversed the basal lamina and connected the distal syncytial tegument to the proximal cell bodies. The tegument contained large numbers of membrane-bound vesicles, especially lined up near the outer surface of the parasite, thus indicating a very active secreting surface. At least two morphological types of membr~e-bound vesicles were identi~ed in the tegument: elongate vesicles of 150-200 X 3050 nm in size, and circular vesicles with a diameter of 150-200 nm. The interior of both types of vesicles usually contained a core with strands of intermediate electron dense material, and a periphery with a more electron dense substance. Elongate vesicles were most frequently observed in the distal part of the tegument, whereas the frequency of circular vesicles was highest further interior. Membrane-bound vesicles of various sizes were furthermore often observed exterior to the surface of the parasite (Figs. 3b, c and d). These vesicles appeared too small to be merely sections through the microvilli without the stalks appearing in the plane of section, and they may represent material released from the parasite surface.
FIG. 3. Transmission electron microscopy of the surface of 2%day old adult E. caproni. (a) Overview of the tegument showing tegumental cytoplasm with organelles (TC) and spine (S), basal Iamina (BL) and microvilli (MV). Scale bar = 5 pm. (b) Microvilli on the distal part of the tegument showing plasma membrane (PM), glycocalyx (GX), elongate vesicles (EV) and extracellular vesicles (XV). Scale bar = 200 MI. (c) Microvilli on distal part of the tegument showing plasma membrane (PM), glycocalyx (GX), elongate vesicles (EV), circular vesicles (CV) and extracellular vesicles (XV). Scale bar = 200 nm. (d) Distal part of the tegument. MicrovilIi densely packed with elongate (EV) and circular (CV) vesicles. Scale bar = 200 nm.
E. caproni surface antigens
115
K.
116
ANDRESEN,
P. E.
SIMONSEN, B.
J.
Parasites recovered 25
4. Recovery of E. caproni from the intestines of mice immunized with detergent solubilized adult surface antigens (fraction 4) and subsequently infected orally with 25 metacercariae. Histo)?;ram indicates mean f S.D. (a) Control group, without keund’s complete adjuvant ‘@CA). (b) FIG.
Control group, with FCA. (c) Experimental group, immunized with antigen fraction 4 + FCA.
IrnrnLi~i~~t~o~with q&ace antigens An attempt was made to immunize mice against reinfection with metacercariae of E. caproni by using adult detergent solubilized surface antigen (antigen fraction 4). Ten mice were immunized with antigen in adjuvant, five control mice received adjuvant only, whereas five control mice did not receive any treatment at all. Four weeks after the first i~unization, all mice were infected orally with 25 metacercariae of E. eapruni. The results are shown in Fig. 4. No resistance to a subsequent infection with E. caproni metacercariae was observed in the immunized mice. DISCUSSION Infections with E. cuproni in mice induce a serum antibody response to the surface of the parasites (Simonsen & Andersen, 1986). However, the antibodies are rapidly lost from the live parasite surface during culturing in vitro, and it has been suggested that this could be due to shedding of surface antigens (Simonsen & Andersen, 1986). The present study demonstrated that antigens are indeed released from the adult E. caproni surface during in vitro culture. Furthermore, all excretory/ secretory antigens recovered from adults had their origin on the worm surface, since they were ail detected in the detergent solubilized surface-antigen fractions as well. If antigens are also released from the intestine or other structures of the parasites, they must be similar to the surface antigens, or they are released in quantities too low to be detected with the employed methods. Four major surface antigens were observed
ANDERSEN
and A.
BIRCH-ANDERSEN
in adult worms, i.e. antigens that consistently showed dense and clear bands in all electrophoretic experiments performed. Additional weaker bands were frequently observed, and these we believe represent minor adult surface antigens. Juvenile antigen fractions revealed only two very weak bands in the electrophoretic experiments, probably due to low antigen concentrations. One antigen was observed in the detergent solubilized fraction only, indicating that this antigen originated on the juvenile surface. Furthermore, this antigen was similar in molecular mass to one of the major adult antigens. Another band observed with the juvenile in vitro antigen fraction corresponded to another of the major adult surface antigens, and therefore probably also originated on the juvenile surface. Two surface antigens were therefore demonstrated on juvenile El. cuproni, both similar in electrophoretic pattern to adult major surface antigens, and at least one of these was released from the juveniles during in vitro culture. The shedding of antigens from the surface of E. caproni may explain the previously described loss of antibodies and attacking cells from the surface of these parasites during in vitro culture (Simonsen B Andersen, 1986). Furthermore, the ability of adult E. cuproni from primary infections to survive for long periods in i~une mice (Sirag et al., 1980; Christensen et al., 1984) may be due to antigen shedding, thus rendering the worms inaccessible to the hosts immune system. The phenomenon of surface antigen shedding during in vitro culture has also been reported for the trematodes F. hepatica (see Hanna, 1980; Lammas Cyr. Duffus, 1983) and S. mansoni (see Kusel, Mackenzie & McLaren, 1975; Wilson & Barnes, 1977; Samuelson, Sher & Caulfield, 1980; Ruppel & McLaren, 1986), and there is accumula~ng evidence that antigen shedding in these parasites is occurring in vivo as well (Burden, Bland, Hammet & Hughes, 1983; Pearce, Basch & Sher, 1986; Ruppel & McLaren, 1986), as an adaptation to withstand the hosts immune attack. The in vitro turn-over rate of E. caproni surface antigens was found by our modified ELISA to be very high, with a half-life of 8-15 min for both adult and juvenile parasites. Furtherm~)re, a control study on juvenile parasites indicated that all surface antigens were released within 45 min. Previous observations on the loss of surface-bound antibodies from juvenile E. caproni, using immunofluorescence microscopy (IFAT), had indicated a slower rate of release, leaving 4-5 h before all antibody was lost from the surface (Simonsen & Andersen, 1986). This discrepancy may be due to different culturing conditions as well as to different sensitivities of the techniques emptoyed in the two studies. The ELISA studies supported the observations from previous IFAT studies (Simonsen & Andersen, 1986) that the surface of adult E. cuproni is covered with antibody when the worms are removed from the
117
E. caproni surface antigens
mouse intestine 4 weeks post primary infection. Since adult E. cuproni from primary infections are not expelled, or otherwise seem to be affected by the hosts immune system at 4 weeks after infection, the antibodies against the surface do not seem to have an immediate effect on the adult worms in viva. Continued release of antigens from the E. cuproni surface in vivo will cause a significant antigenic stimulus to the host. However, immunization of mice with detergent solubilized surface antigens from adult worms in the present study did not lead to any reduction in the worm establishment in a subsequent oral infection with metacercariae of E. cuproni. This failure may be related to the immunization procedure and to the nature of the antigen. Since the parasites are purely intestinal, the antigens may need local intestinal presentation (Befus & Bienenstock, 1982) instead of subcutaneous or intraperitoneal injection. Furthermore, antigens produced from juvenile parasites may be more potent stimulators of resistance, since it is known from previous experiments that resistance is directed against the juvenile parasites (Christensen et al., 1986). Similar immunization of rodents with excretory/secretory products of Fusciola heputicu (see Lehner & Sewell, 1979; Rajasekariah, Mitchell, Chapman & Montague, 1979; Burden, Harness & Hammet, 1982) have generally yielded better results when antigens were derived from juvenile or young flukes than from adults. In the present study it was not possible to produce sufficient amounts of juvenile antigen for this purpose. Resistance-related factors such as reduction in growth or reproductive capacity or reduced pathology due to infection were not examined in the present investigation, but may well have been affected by the immunization. The EM studies on the surface of adult E. caproni revealed a highly active tegument, and thereby reflected the high rate of antigen shedding from the parasite surface. In gross structure, the tegument was similar to what has been described from F. hepaticu (see Threadgold, 1963, 1967, 1984), with a distal syncytial cytoplasm containing organelles and spines, and limited outwards by a trilaminate plasma membrane covered by a thin glycocalyx. The tegument was densely packed with membrane bound vesicles of at least two morphological types, which were lined up along the outer membrane, the elongate vesicles usually perpendicular to the membrane. It is tempting to suggest that these vesicles contain antigenic material which is released onto the parasite surface, thereby maintaining the continued antigen shedding. Small membrane-bound vesicles were furthermore observed outside the parasite. These vesicles may contain material released from the parasite surface. More detailed ultrastructural studies on the dynamics of the E. cuproni tegument are in progress. Acknowledgements-The following are valuable assistance and discussions during
thanked for the course of
these investigations: Dr Palle H. Jakobsen and his staff in the Department of Treponematosis, Statens Serum Institut, Mrs Helene Ravn, Department of Biophysics, Statens Serum Institut, Dr Jerrn Andreassen, Institute of Population Biology, University of Copenhagen, and Dr Niels 0. Christensen, Danish Bilharziasis Laboratory. The investigations were supported by grants from the Danish Natural Science Research Council.
REFERENCES BEFUS D. & BIENENSTOCK J. 1982. Factors involved in symbiosis and host resistance at the mucosal-parasite interface. Progress in Allerav 31: 76-177. BURDEN D. J., I&NESS E. ~‘HAMMET N. C. 1982. Fasciola hepafica: attempts to immunize rats and mice with metabolic and somatic antigens derived from juvenile flukes. Veterinay Parasitology 9: 26 l-266. BURDEN D. J., BLAND A. P., HAMMET N. C. & HUGHES D. L. 1983. Fasciola heuafica: mieration of newlv excysted juveniles in resistant rats. Experimental Parasitology 56: 277-288. BUTT~RWORTHA. E., TAYLOR D. W., VEITH M. C., VADAS M. A., DESSEIN A., STURROCKR. F. & WELLS E. 1982. Studies on the mechanisms of immunity in human schistosomiasis. Immunological Reviews 61: 5-39. CHRISTENSEN N. 0., NYDAL R., FKANDSEN F. & NANSEN P. I98 I. Homologous immunotolerance and decreased resistance to Schistosoma mansoni in Echinostoma revolutum-infected mice. Journal of Parasirology 61: 164-166. CHRISTENSEN N. 0.. FAGBEMI B. 0. & NANSEN P. 1984. TTpanosoma brucei-induced blockage of expulsion of Echinosloma revolutum and of homologous E. revoluturn resistance in mice. Journal ofParusitology 70: 55% 561. CHRISTENSEN N. O., KNUDSEN J. & ANDREASSEN J. 1986. Echinostoma revolulum: resistance to secondary and superimposed infections in mice. Experimental Parasitology61:311-318. HANNA R. E. B. 1980. Fasciola hepatica: glycocalyx replacement in the juvenile as a possible mechanism for protection against host immunity. . Experimental Para. &ology 50: io3- i 14. HUGHES D. L. 1985. Trematodes, excluding schistosomes with special emphasis on Fasciola. In: Parasite Antigens in Prote&ion, LIia&osis and Escape (Edited by PARKHOUSE R. M. E.). Currem Topics in Microbiology and Immunology 120: 24 I-260. JAKOBSEN P. H., THEANDER T. G., JENSEN J. B., MBLBAK K. & J~PSEN S. 1987. Soluble Plasmodium falciparum antigens contain carbohydrate moieties important for immune reactivity. Journal of Clinical Microbiolog?, 25: 2075-2079. JEPSEN S. & AXELSEN N. H. 1980. Antigens and antibodies in Plasmodium falciparum malaria studied by the immunoelectrophoretic method. Acta Pathologica, Microbiologica et Immunologica Scandinavica, Section C 88: 263-270. KUSEL J. R., MACKENZIE P. E. &MCLAREN D. J. 197.5.The release of membrane antigens into culture by adult Schistosoma mansoni. Parasitology 11: 247-259. KYHSE-ANDERSEN J. 1984. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer I
ll8
K.
ANDRESEN,
of proteins from po~yac~~~de
P. E.
SZMONSEN,B.
J.
to &trocefIulose. Joldrnai
~f~~~c~emical and 3jop~ys~cul Methods 10: 203-209. LAEMMLI U. K.
1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: 680-685. LAMMASD. A. & DUFFUSW. P. H. 1983. The shedding of the outer glycocatyx of juvenile Fasciola hepatica. Veterinary Parasitology 12: 165-l 78.
LEHN~R R. P. & SEWELL M. M.H. 1979. Attempted immunisation of laboratory animals with metabolic antigens of Fusciofn ~epat~~a. Veterinary Science Communi~a~~~~ 2: 337-340.
MCLAREND. J. 1980. Schistosoma mansoni: The Parasite Surface in Relation to Host Immunity. Research Studies Press, John Wiley, New York. ODAIBOA. B., CHRISTENSEN N. 0. & UKCILIF. M. A. 1988. Establishment, survival and fecundity in Echinosfoma caproni infections in NMRI mice. Proceedinns of the H~lm~~thol~~ical Society of Wash~~~t~~~ 55: 26<-289. PEMCE E. J.. BASCH P. F. & SEER A. 1986. Evidence that the r&u&d surface antigenicity of developing fch&osoma mansoni schistosomula is due to antigen shedding rather than host molecule acquisition. Parasite Immunology 8: 79-94.
RAJASEKARIA~ G. R., MITCHELLG. F., CHAPMANC. B. & MONTAGUEP. E. 1979. Fasciola hepatica: attempts to induce orotection against infection in rats and mice by injection of excreto$secretory products of immature worms. Parasitolo~ 79: 393-400. REDDINGTON J. J., Lim R. W. & WEXOTT R. B. 1984. A review of the antigens of Fasciola hepatica. ~ter~~a~ Par~itolo~ 14: 209-229. RUPPEL A. & MCLAREN D. J. 1986. Schktosoma mansoni:
ANDERSEN
and A. BIRCH-ANDERSEN
surface membrane stability tn vitro and in v&o. Experimen~ul Parusitology $2: 223-236. SAMUELSON J. C., SHERA. & CMJLPIELD.i. P. 1980. Newly transformed schistosomula spontaneously lose surface antigens and C3 acceptor sites during culture. Journal of Immunology 124: 20552057. SIMONSEN P. E. & ANDERSENB. J. 1986. Echinostoma rev&turn in mice; dynamics of the antibody attack to the surface of an intestinal trematode. ~~e~~~r~o~a~Journal .~orPff~~ol~~ 16: 475-482, SIMPS~~~A. 3. G. C SYIJTHERS S. R. 1985. Schistosomes: surface, egg and circulating antigens. In: Parasite Antigens in Protection, Diagnosis and Escape (Edited by PARK~ONSB R. M. E.). Current Topics in Microbiology and Immunology 120: 205-239. &RAG S. EL,?IHRISTENSEN N. Is)., FRANDSENF., MONRAD J. & NANSENP. 1980. Homologous and heterologous resistance in Echinosroma revo&um infections in mice, Parasitolo~
80: 479-486.
THREADGOLD LT. 1963. The tegument and associated structures of Firs&la hepatica. quarterly Jourzal of microscopical Science 104: SQ5-5 12. THPEALYGOLDL.
T. 1967. Electron microscope studies of Further observations on the tegument and associated structures. Parasitology 57: Fasciola
hepatica-III.
633-637.
THREADGOLDL. T.
1984. Parasitic platyhelminths. In: Vol. 1 (Ehiteh by BEREITERHAHN J.. M~TOLTS~A. G. & RICHARDS K. S.f., I. DV. 13219 1. Springer, Berlin. WILSON R. A. & BAENE~ P. E. 1977. The formation and turnover of the membr~acalyx on the tegument of Biology of the fntegument.
Schistosoma mansoni. Parasitology 74: 6 i-7 1.