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Parasitol Res (1989) 75:343-347
9 Springer-Verlag 1989
Characterization of a surface antigen
of Eimeria nieschulzi (Apicomplexa, Eimeriidae) sporozoites
Stanislas Tomavo 1, Jean-Francois Dubremetz t, and Rolf Entzeroth 2
1 Inserm, U42, Certia, 369, rue Jules - Guesde, 59650 Villeneuve - D'Ascq, France
z Zoologisches Institut der Universit/it, Poppeldorfer Schloss, D-5300 Bonn 1, Federal Republic of Germany
Abstract. A monoclonal antibody (McAb 3C3)
reacting with a pellicular antigen of Eimeria nieschulzi sporozoites has been selected among hybridomas produced against this organism by immunofluorescence assay. This antigen has been shown
to be located on the zoite surface by immunofluorescence on living organisms. Capping and
shedding of antigen-monoclonal antibody immune
complexes was observed upon incubation at 37 ~ C.
On western immunoblotting, two polypeptides at
22 and 26 kDa were recognized by McAb 3C3,
whereas only one polypeptide of 22 kDa was immunoprecipitated by the same antibody after lactoperoxidase surface radio-iodination of sporozoites.
Coccidiosis is a complex intestinal disease caused
by Eimeria species. Both humoral and celt-mediated immune reactions have been implicated in
acquired immunity to coccidiosis (Long and Jeffers
1986). The goal of recent research is to identify
parasite antigens that elicit protective immunity in
the host; surface antigens of Eimeria sporozoites
have been found to be prominent candidates
(Brothers etal. 1988; Clarke etal. 1987; Crane
et al. 1988; Gore and Newman 1986).
During processes of recognition, invasion, and
early development, parasite-host cell relationships
play a key role in the successful infection of the
host. Surface antigens are most likely to be involved during parasite-host cell contact and the
interiorization of coccidian parasites (Augustine
and Danforth 1985). In the present paper, we report on the characterization of a surface antigen
of the rat coccidium E. nieschulzi and the interacReprint requests to: J.-F. Dubremetz
tion of this antigen with a specific monoclonal antibody on living sporozoites.
Materials and methods
Monoclonal antibody production. Monoclonal antibodies (mcab)
were obtained by the fusion of SP2/O myeloma cells with splenocytes of BALB/e mice immunized i.p. with sporozoites of E.
nieschulzi emulsified in Freund's complete adjuvant. After two
repeated immunizations 3 weeks apart with incomplete
Freund's adjuvant, mice were boosted i.v. with 2 x 106 purified
sporozoites lysed by freeze-thawing. The fusion was carried out
3 days later according to the method of Galfre et al. (1977).
Screening of hybridomas was done by immunofluorescence assay and Western blotting; positive hybridomas were cloned by
limit dilution. Mass production of mcabs was accomplished
by the injection of hybridoma cells into pristan-primed BALB/c
mice and subsequent collection of ascitic fluids. Only one of
the mcabs obtained (3C3) is described here.
Surface iodination of parasites. Purified sporozoites of E. nieschulzi were surface-labelled with 125I by lactoperoxidase-catalyzed radioiodination (Marchalonis etat. 1971). A total of
10 x 106 sporozoites were resuspended in phosphate-buffered
saline (PBS : 150 mM NaC1, 50 mM sodium phosphate buffer;
pH 7.4)containing 10 gl/ml 12SI-sodium (3.7109 Bq/ml, carrierfree; Amersham), 5 gg/ml lactoperoxidase (Calbiochem,
100 IU/mg), 1.25 gM KI, and 40 gM H202. This suspension
was incubated at 4~ C for 10 min with mild agitation. Labelling
was stopped by adjusting the suspension to 30 mM NAN3. Labelled sporozoites were then washed twice with PBS and processed for immunoprecipitation.
[mmunoprecipitation. Radioiodinated sporozoites were resuspended in a lysis solution containing 0.5% (v/v) Nonidet P40
(NP 40), 2 mM ethylene diamine tetraacetic acid, 10 mM KI,
1 mM phenyl methyl sulfonyl fluoride, and 0.1 mg/ml aprotinin
in PBS and incubated for 1 h at 4~ C. The suspension was then
centrifuged at 10000 g for 15 min and the supernatant fraction
was used for immunoprecipitation. Aliquots of the lysates were
taken for radioactivity measurement after trichloroacetic precipitation of proteins.
Immune complexes were prepared by incubating the equivalent of 100000 counts/min (cpm) lysate with 20 ~1 ascitic fluid
in i ml lysis buffer for 4 h at 4~ in a rotary shaker. The
immune complexes were then purified by overnight incubation
344
S. Tomavo et al. : Eimeria surface antigen
at 4 ~ on 20 lal immunosorbent prepared with rabbit antimouse IgG immunoglobulins absorbed on Protein A-Sepharose
4B (Pharmacia). The gel was washed five times with a washing
buffer containing 1 M NaC1, 0.5% NP 40, and 10 m M KI in
TRIS-HC1 buffer (pH 8.3) and then eluted with electrophoresis
sample buffer.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) was carried out was according to the method of
Laemmli (1970), using 12% (w/v) acrylamide separating gels.
Molecular weight standards (Pharmacia, LMW kit) were used
for calibration. Gels were stained with Coomassie blue R, dried,
and exposed to Kodak X-O M A T A R film at - 8 0 ~
with
Cronex Lightning plus intensifying screens (DuPont).
Western blotting. Purified sporozoites were separated by SDSP A G E ' u n d e r nonreducing conditions (10 v sporozoites per
1-cm-wide slot) and electrophoretically transferred to nitrocellulose at 200 mA (1 h) according to the technique of Towbin
et al. (1978). The nitrocellulose sheet was then saturated for
30 min in 5% nonfat dry milk in buffer (140 m M NaC1 and
0.5% (v/v) Tween-20 in 15 m M TRIS-HC1; pH 8 : TNT). Strips
were then cut and incubated with the various mcab solutions
(mouse ascitic fluid diluted 1:200 in TNT) for 1 h at 37 ~ C.
After washing, the sheet was incubated in peroxidase-conjugated anti-mouse IgG serum (Nordic) diluted in TNT and developed with diaminobenzidine.
Immunofluorescence assay. For hybridoma screening, purified
sporozoites were washed three times with PBS and dried on
standard immunofluorescence assay (IFA) slides, which were
stored at - 2 0 ~ C. I F A was carried out at 37 ~ C after a 10-rain
fixation in cold acetone. For surface fluorescence, purified tachyzoites were used either alive or after a 5-min fixation with
2% (v/v) formalin in PBS; they were incubated in fluoresceinconjugated rabbit anti-mouse IgG antibodies, all steps being
carried out at 4 ~ C.
Light and electron microscopy. For studying surface antigen
before and after invasion, E. nieschulzi sporozoites were incubated in 3C3 ascitic fluid for 15 rain, washed in PBS suspended
in Minimum Essential Medium (MEM), inoculated onto monolayers of 3T3 cells at a density of 10 6 sporozoites/cm z, and
incubated at 37 ~ C. Indirect immunodetection techniques were
used in this study. For immunofluorescence, fluorescein-conjugated rabbit anti-mouse IgG immunoglobulins diluted 1:50
were used. For electron microscopy, peroxidase-conjugated
goat anti-mouse IgG Fab fragments (Nordic) were also diluted
1:50. All dilutions and washes were done in PBS (pH 7.4);
mcab and immune sera were diluted 1 : 25.
Immunofluorescence was carried out on either living sporozoites or sporozoites or cells fixed for 30 rain in 3% formalin
in PBS. For the detection of intracellular sporozoites, fixed
monolayers were treated with cold acetone (Lazarides and
Weber 1974). When the sporozoites had been preincubated with
mcab before interaction with the cells, this step was omitted
after fixation. Observations were done using a Leitz Orthoplan
microscope equipped for epifluorescence; pictures were taken
on Kodak Ektachrome 400 film.
For immunoperoxidase detection, the monolayers were
fixed with paraformaldehyde picric acid (Stephanini et al. 1967)
for I h at 4 ~ C, followed by overnight washing in PBS: cells
were then equilibrated for 1 h in 10% glycerol in buffered saline
and were frozen in liquid nitrogen for 10 s. The specimens were
then successively incubated in normal goat serum diluted 1 : 30
in PBS, mcab 3C3, and peroxidase conjugate for 30 rain, with
Fig. 1. (a) Immunoblot of SDS-Page total E. nieschulzi sporozoite proteins probed with mcab 3C3 under reduced (+DDT)
conditions. (b) Immunoblot of SDS-Page total E. nieschulzi
sporozoite proteins probed with mcab 3C3 under nonreduced
( - D D T ) conditions. (c) Autoradiogram of total NP-40 extract
of E. niesehulzi sporozoites after surface 112s iodination. (d)
Autoradiogram of E. nieschulzi iodinated surface proteins after
immunoprecipitation with mcab 3C3
extensive washing in PBS between each step. When the sporozoites had been pretreated with mcab 3C3 before the interaction, this step was omitted after fixation. The coverslips were
then fixed with 1% glutaraldehyde in PBS for 10min and
washed in buffered saline for 30 min. The peroxidase was revealed by a 3-rain incubation in 0.05% diaminobenzidine in
0.1 M TRIS-HC1 buffer (pH 7.4, 0.006% H2Oz). After washing, the monolayers were postfixed in 2% OsO~ in 0.1 M phosphate buffer (pH 7.4) for 2 h at 4 ~ C, dehydrated, and embedded in Epon. After polymerization, parasite-rich areas were
selected, punched off the plastic sheet, glued on an Epon block,
and sectioned parallel to the plane of the monolayer. Unstained
S. Tomavo et al. : Eimeria surface antigen
345
Fig. 2. Indirect immunofluorescentlight micrograph of live E. nieschulzi sporozoites after incubation with mcab 3C3 and labeling
with anti-mouseFtTC. Note the differentdegree of immune-complexcapping, x 760. Fig. 3. Electronmicrograph of an E. niesehulzi
sporozoite after incubation with mcab 3C3 and labeling with peroxidase conjugate. The entire surface of the parasite is labeled.
The beginning of immune-complexcapping is indicated by a few electron-dense vesicles (arrows). ap, apical pole; rh, rhoptries;
pp, posterior pole; rb, refractile body, x 8350. Fig. 4. Higher magnificationof the posterior pole (pp) of a sporozoite, showing
the partially labeled surface interrupted by nonlabeled plaques (arrows). Note the accumulation of capped vesicular immune
complexes (es). x 23750
thin sections were observed with a HitachiU 12 electronmicroscope.
Results
The 3C3 mcab was tested on Western blots of E.
nieschulzi sporozoite proteins after electrotransfer
from SDS-PAGE. Sporozoite antigens were tested
under reduced (Fig. 1, a) and non-reduced conditions (Fig. 1, b). Two antigens (22 and 26 kDa)
reacted specifically on an immunoblot under reduced conditions; the 22-kDa band stained more
intensely than the 26-kDa band (Fig. 1, a). Under
nonreduced conditions, two major bands of equal
intensity were observed at 22 and 24 kDa (Fig. 1,
b). Surface iodination of Nonidet P 40 extracts of
sporozoites followed by SDS-PAGE analysis
under reduced conditions showed the existence of
four major molecules of 66, 26, 22, and 16 kDa
(Fig. 1, c). On immunoprecipitation of this extract,
mcab 3C3 reacted only with the 22-kDa band
(Fig. 1, b).
Light and electron microscopy
Live sporozoites of E. nieschulzi, incubated with
mcab 3C3 and labeled with anti-mouse fluorescein
isothiocyanate (FITC), showed a bright surface
fluorescence (Fig. 2, lower right). However, at
10-20 rain after incubation, live sporozoites had
capped immune complexes at their posterior pole
(Fig. 2); this was followed by shedding of the complexes, resulting in nonlabelled, live sporozoites.
Electron micrographs of sporozoites incubated
with mcab 3C3 before the invasion of host cells
showed a homogeneous surface labelling after incubation with peroxidase conjugate and revelation
with diaminobenzidine (Fig. 3). Internal organelles
[rhoptries (rh), micronemes (ran), refractile bodies
(rb)] were not affected by the incubation procedure. After the incubation of live sporozoites with
mcab 3C3 for 15 min, different stages of surfaceimmune-complex capping were revealed by electron microscopy. Single as well as clustered vesicles
with an electron-lucent inner core and electron-
346
dense periphery and measuring 10-15 nm were
shed from the surface of the sporozoites (Fig. 3).
The formerly complete electron-dense, labelled
surface of the parasites showed patches of nonlabelled membrane (Fig. 4, arrows). Finally, the labelled surface immune complexes were shed at the
posterior pole of the sporozoite (Fig. 4).
Discussion
From its reactivity by IFA with the surface of live
E. nieschulzi sporozoites, we conclude that mcab
3C3 recognizes a surface antigen on these organisms. This was also confirmed by immunoprecipitation data, since mcab 3C3 immunoprecipitates
a radioactive protein comigrating with polypeptide
found by iodinating sporozoites via the lactoperoxidase procedure. When electrophoresed under reducing conditions, this antigen has a mol.wt, of
22 kDa after iodination.
The reaction of mcab 3C3 on Western blots
was more complex, as two bands were identified
on gels containing reduced or non-reduced sporozoite proteins; this may be due either to the fact
that 3C3 would recognize an epitope common t o
two antigens, one of which would not be on the
sporozoite surface, or to the existence of a 26-kDa
intracellular precursor of the 22-kDa surface antigen.
The four surface antigens of E. nieschulzi
sporozoites were similar in molecular weight to the
surface antigens of E. acervulina, E. maxima, E.
tenella, and E. nieschulzi previously described by
Wisher (1986). The capping phenomenon has been
described in coccidian Sporozoites for several ligands (Russell and Sinden 1981; Augustine and
Danforth 1982), and the capping of mcab immune
complexes by coccidian sporozoites has been described for E. tenella (Speer et al. 1983a, 1983b,
1985). These authors showed that a secondary conjugate was needed for the capping of mcab on the
sporozoite surface, which was also the case in the
present study. Capping of mcabs has also been
found in Toxoplasma gondii (Dubremetz et al.
1985), but in that case capping and shedding occurred only during host cell invasion. In both
cases, however, immunolabelled vesicles accumu,
lated on the posterior extremity of the sporozoites,
which was also the case with E. nieschulzi in the
present study and indicates that ligands are associated with the capping of antigens. We did not
determine whether the antigen against which mcab
3C3 reacts remained on the surface of the sporozoites after capping and shedding of the immune
S. Tomavo et al. : Eimeria surface antigen
complexes. Further study is needed to elucidate
how these organisms modulate the interaction of
their surface molecules with antibodies. It is not
clear whether immune-complex capping reflects a
mechanism by which the parasite evades the hostcell immune response (resembling the shedding of
CS-protein in Plasmodium spp. ; Cochrane et al.
1976) or represents a phenomenon of membrane
turnover and fluidity related to cell motility (Heath
1983; Bretscher 1984).
Acknowledgements. The authors thank C. Ansel and M. Mortuaire for excellent technical assistance. This study was funded
by INSERM and a stipend from the Deutsche Forschungsgemeinschaft (DFG) to R. Entzeroth.
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Accepted October 1, 1988