Article available at http://www.parasite-journal.org or http://dx.doi.org/10.1051/parasite/2004112225
IMMUNOLOCALIZATION OF AN ENTEROTOXIC GLYCOPROTEIN EXOANTIGEN
ON THE SECRETORY ORGANELLES OF CRYPTOSPORIDIUM PARVUM SPOROZOITES
E L - S H E W Y K.A.* & EID R.A.**
Summary:
Résumé
In this study, the fine ultrastructures of the secretory organelles of
C. parvum sporozoites were demonstrated using transmission
electron microscopy (TEM). Meanwhile, a previously identified
enterotoxic 1 8-20 kDa copro-antigen (1 8-20 kDa CCA),
associated with cryptosporidiosis in both human and calves, was
isolated and immunolocalized on C. parvum sporozoites. Using
immunoelectron microscopy and anti-18-20 kDa monospecific
antibody demonstrated marked existence of the 18-20 kDa CCA
on the apical organelles and at the trilaminar pellicles. An anterior
extrusion of this protein was demonstrated around the excysted
and released sporozoites. However, non excysted sporozoites did
not show this protein. Affinity blotting, with biotinylated jacalin,
demonstrated the O-linked oligosaccharide moiety of this protein.
The potential role of this protein in the host cell invasion and/or
gliding motility remains unelucidated. However, its enterotoxicity,
location and secretory nature suggest that it may be a target for
neutralization or invasion inhibition of Cryptosporidium.
ENTÉKOTOXIQUE DANS LES ORGANITES SÉCRÉTOIRES DE SPOROZOITES DE
: IMMUNOLOCALISATION D'UN EXOANTIGÈNE
CRYPTOSPORIMI
fine des organites
de C. Parvum a été étudiée en
électronique
entérotoxique
à transmission
(TEM),
de sporozoïte
(18
bovins,
était localisé
alors qu'un
monospécifique
l'antigène
trilaminaire.
repérable
anti-antigène
dans les organites
Une extrusion
(18-20
kDa)
révèle la présence de
biotinylée
alors
Le
a révélé
Le rôle
de la cellule et dans le
n'a pas été tiré au clair. Cependant,
sa localisation
et sa nature sécrétoire
qu'elle pourrait être la cible d'une neutralisation
de l'invasion
est
non dékystés.
O liée sur cette protéine.
potentiel de cette protéine dans l'invasion
du sporozoïte
pellicule
relaché par dékystement,
réalisé avec de la jacaline
une partie oligosaccharidique
glissement
de l'homme ou des
L'anticorps
apicaux et dans la
qu'elle n'existe pas autour des sporozoites
transfert d'affinité
préalablement
antérieure de celte protéine
autour du sporozoïte
de
coproantigène
kDa CCA),
à l'aide d'anticorps.
sécrétoires
microscopie
identifié et associé avec la cryptosporidiose
entérotoxicité,
KEY W O R D S : Cryptosporidium parvum. coproantigen, 1 8-20 kDa
glycoprotein, exoantigen, immunolocalization.
M PARVU M
Dans cette étude, l'ultrastructure
sporozoites
GLYCOPROTÉIQUE
son
suggèrent
ou d'une
inhibition
de Cryptosporidium.
MOTS CLÉS : Cryptosporidium parvum, coproantigène, glycoprotéine de 1820 kDa, exoantigène, immunolocalisation.
C ryptosporidium
parvum (C. parvum)
is an apic o m p l e x a n protozoan parasite that is increasingly recognized as a major cause of diarrhea
and gastroenteritis in both human and animals (Laurent et al., 1 9 9 9 ) . In the past few years the parasite
received a great deal o f attention b e c a u s e o f the persistent fatal diarrhea in the i m m u n o c o m p r o m i z e d
patients, and b e c a u s e o f the public health threat of
water b o r n e outbreak in otherwise healthy individuals
(Mackenzie et al., 1 9 9 5 ) . So far, no approved effective
drug or immunotherapy for controlling or prevention
* Microbiology a n d Parasitology Department, Faculty o f Medicine.
S u e z Canal University, Ismailia, E g y p t .
** E l e c t r o n M i c r o s c o p y C e n t e r , F a c u l t y o f S c i e n c e , Z a g a z i g U n i v e r sity, Egypt.
C o r r e s p o n d e n c e : D r K h a l i d A. E l - S h e w y , M i c r o b i o l o g y D e p a r t m e n t .
C o l l e g e o f M e d i c i n e a n d M e d i c a l S c i e n c e s , K i n g K h a l i d University,
P O B o x 6 4 1 , A b h a . K i n g d o m o f Saudi A r a b i a . F a x : 0 0 9 6 6 7 2 2 4 7 5 7 0 .
E-mail:
[email protected]
of cryptosporidiosis is currently available (Fayer &
Ungar, 1986). T h e invasive stages o f apicomplexan
parasites characteristically possess sets o f secretory
organelles. T h e s e organelles proved to b e involved in
the production and storage o f secretory materials that
assist in gliding motility, cells adhesion and host cell
invasion (Lingelbach & Joiner, 1998; Langer & Riggs,
1999; Preiser et al., 2 0 0 0 ) . Many workers refer to the
importance of such secretory products in developing
vaccines against apicomplexan parasites as
Plasmodia,
Toxoplasma
and Eimeria (Schwanzman 1986; Whitmire
et al., 1988; Perkins, 1 9 9 2 ) .
In a previous study w e have identified and isolated an
18-20 kDa Cryptosporidium
copro-antigen (18-20 kDa
CCA), which demonstrated an enterotoxic activity (ElShewy et al., 1994; El-Shewy et al., 1999). In the present study w e have attempted to demonstrate the
ultrastructures o f the sporozoites secretory organelles
and to localize this protein using a m o n o s p e c i f i c anti18-20 kDa antibody.
225
MATERIALS AND METHODS
STOOL SAMPLES COLLECTION
AND ANTIGEN PREPARATION
P
ositive stool samples o f C. parvum
(as proved
by microscopic examinations) were collected from
naturally infected dairy calves. Stool samples
w e r e used in purification and isolation of C.
parvum
oocysts and as a source for extraction of the previously
detected 18-20 kDa protein (El-Shewy et al., 1 9 9 4 ) .
Sodium dodecylysulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroelution techniques
w e r e used to analyze and extract the protein band of
interest ( 1 8 - 2 0 k D a ) according to Laemmli ( 1 9 7 0 ) and
J a c o b s & Clad ( 1 9 8 6 ) respectively.
OOCYSTS PURIFICATION AND EXCYSTATION
C. parvum
oocysts w e r e concentrated and purified
from stool samples using Sheather's floatation and
cesium chloride (CsCl) gradient methods (Ma & Soave,
1983; Kilani & Sekla, 1985). T h e concentrated purified
C. parvum
oocysts w e r e excysted with the m e t h o d o f
Current & Haynes ( 1 9 8 4 ) .
ANTI 1 8 - 2 0 K D A MONOSPECIFIC ANTIBODY
PREPARATION
Pathogen free 2.5 kg New Zealand White (NZW) female
rabbits w e r e used to raise m o n o s p e c i f i c antibody
against the electroeluted 18-20 kDa protein. Rabbits
received four b o o s t e r doses of 125 ug purified and
c o n c e n t r a t e d protein mixed with 1 ml of Freund's
incomplete adjuvant, via intramuscular and subcutan e o u s injection at different sites, with ten days intervals. Rabbits w e r e bled before immunization and two
w e e k s after the last injection. Collected sera w e r e
tested against the purified 18-20 kDa protein by immunoblotting ( T o w b i n et al., 1 9 7 9 ) .
ELECTRON MICROSCOPIC STUDIES
Excysted C. parvum sporozoites were aliquoted in 150 µl
volume and mixed with an 8 5 0 µl aliquot o f glutaraldehyde formaldehyde fixative solution throughout processing. T h e samples w e r e p r o c e e d e d for electron
microscopic examination following the method o f Roth
et al., 1981 and Armbruster et al., 1982. Briefly, samples
were centrifuged, washed three times and left in buffer
overnight at 4° C. T h e samples w e r e dehydrated in a
graded series o f alcohol, then sectioned, picked up on
nickel grids and floated o n 1 % b o v i n e serum albumin
( B S A ) in phosphate buffer saline ( P B S ) pH 7.4 for
15 min. T h e y w e r e left to react with a drop o f m o n o specific anti 18-20 kDa antibody, or control rabbit
serum, diluted 1:100 with 1 % BSA in PBS for 30 min
226
at r o o m temperature, then with 0.5 % BSA for 10 min
and further incubation for 30 min with 10 nm goat antirabbit IgG-gold colloidal particles ( E . Y labs Inc. San
Mateo, CA.94401). Samples w e r e w a s h e d and then
stained with 5 % urinyl acetate. T h e y w e r e e x a m i n e d
in a Philips model 4 1 0 electron m i c r o s c o p e at an a c c e lerating potential of 80 kv.
AFFINITY BLOTTING
Twenty five ug of the electroeluted 18-20 kDa protein
was analyzed b y SDS-PAGE and transferred to the
nitrocellulose paper in Tris-glycine-methanol buffer
( T o w b i n et al., 1 9 7 9 ) . Electrophoresis w a s performed
for 30 min at constant current o f 4 0 0 mA, and the blot
was then b l o c k e d with 5 % nonfat milk in T B S (trisbuffered saline; 0.15 M Nacl, 0,05 M tris-Hcl, pH 7.6)
for o n e hour at room temperature. After washing in
TBS, the blot w a s incubated with c o n c a n a v a l i n A
(Pharmacia; 0.005 % w/v in T B S containing 0.0005M Ca
C12, 0.5 % BSA) for two hours at room temperature,
washed again in TBS, and incubated 90 min in 0.005 %
(w/v) horse-radish peroxidase (Sigma T y p e VI). Color
w a s d e v e l o p e d b y addition o f 0.05 % diaminobenzidines tetrahydrochlorid (Sigma) in T B S with H 2 0 2
(0.01 % w/v) for 2-3 min. T h e stained blot was w a s h e d
in water and dried (Scott & Dodd, 1 9 9 0 ) . Alternatively,
s o m e blots w e r e stained with biotinylated jacalin
(Vector Lab, Inc. C o ) according to the supplier's instructions (Rohringer & Holden, 1985).
RESULTS
ULTRASTRUCTURE
U sing T E M . Cryptosporidium
sporozoites demonstrated three layered pellicle, together with an
underlying double unit membrane. T h e conoid
and crystalloid bodies were observed at the anterior and
posterior ends respectively. Meanwhile, sporozoites characteristically demonstrated three sets o f secretory organelles. At their leading e n d two rhoptries and many
narrow rhoptries ducts were demonstrated. Rhoptries
were found with two distinct regions; a basal bulbous
part and narrow apical duct. Small fusiform or flaskshaped micronemes were found in a large number. T h e
third demonstrated vesicles were the dense granules
which were scattered in few numbers in the sporozoites.
All these vesicles appeared as electron dense bodies in
the nonexcysted forms (Fig. 1A & B ) .
IMMUNOLOCALIZATION
C. parvum
oocysts containing non excysted, non relea-
sed sporozoites did not s h o w any internal or external
labelling w h e n they w e r e incubated with monospecific
Fig. 1. - (A) Transmission electron micrograph of intact sporulated
C. parvum oocyst, demonstrating four sporozoites (Sp) with electron dense micronemes (Mn), dense granules (Dg), and membrane
pellicle (Mp); Or, Oocyst residuum; Ow, oocyst wall, x 17.000.
(B) Transmission immunoelectron micrograph of nonexcysted sporozoite demonstrating the conoid (Co): two club shaped rhoptries
( a r r o w beads), and rhoptries ducts (Rd). Micronemes (Mn). dense
granules (Dg). crystalloid body (Cb) and membrane pellicle (Mp).
All these organelles appear as electro-dense structures, x 24,000.
18-20 kDa antibody using immunoelectron microscopy.
O n the other hand, transverse micrographs in the
e x c y s t e d , but unreleased sporozoites demonstrated
minimal labelling at their secretory organelles with no
labelling at their trilaminar pellicles. Meanwhile their
secretory vesicles w e r e found less electron-lucent than
those o f the excysted and completely released sporozoites (Fig. 2A, B, C). Excysted and released C. parvum
sporozoites appeared to have a characteristic labelling
pattern. Gold particles w e r e concentrated principally
at the apical e n d o f the excysted sporozoites as well
as at the trilaminar pellicle. Diffuse labelling was also
detected as anterior extrusion of these sporozoites.
Many electron-lucent vacuoles with variable sizes and
with m e m b r a n e whorls inside w e r e observed a m o n g
the labelled sporozoites (Fig. 3A). Some excysted sporozoites s h o w e d labelling over their entire b o d i e s
(circum-sporozoite) with varying intensity; this was
demonstrated in scanning electron microscopy (Fig. 3B).
Fig. 2. - (A, B & C) r e p r e s e n t different
transverse
immuno-electron
micro-
g r a p h s o f intact e x c y s t e d n o n r e l e a s e d
s p o r o z o i t e r e a c t e d with a n t i - 1 8 - 2 0 k D a
monospecific
antibody,
showing
m i n i m a l s c a t t e r e d g o l d particles. T h e s e
sections
demonstrate
the
fine
ultra-
structures o f t h e Cryptosporidium
rozoite ( S p ) with
less
spo-
electron-dense
micronemes ( M n ) , dense granules
(Dg).
Nn, n u c l e u s ; Sd. s u b p e l l i c u l a r ducts;
conoid, and membrane
pellicle
Co,
(Mp).
x 24,000.
227
Fig. 3 - ( A ) I m m u n o e l e c t r o n
of excysted
C. parvum
and
micrograph
completely
released
s p o r o z o i t e , r e a c t e d with m o n o -
specific anti-18-20 kDa antibody, d e m o n s trating g o l d
c o n o i d (Co)
particles
clustered
on
the
c o n c e n t r a t e d a n d distributed
o v e r t h e m e m b r a n e p e l l i c l e (Mp)
a n d for-
m i n g a trail in front o f t h e r e l e a s e d s p o rozoites (S). Note electron-lucent vacuole
(V)
and
micronemes
(Mn),
with
many
whorls, x 24.000.
( B ) Scanning immunoelectron micrograph
o f C. parvum
excysted and released spo-
rozoite, r e a c t e d with m o n o s p e c i f i c a n t i - 1 8 20 kDa antibody, s h o w i n g gold particles
coating the entire sporozoite surface (circ u m s p o r o z o i t e ) , a n d in front o f t h e s p o r o z o i t e (S), x 1 1 . 0 0 0 .
AFFINITY' BLOTTING
Affinity staining o f the 18-20 kDa protein revealed the
p r e s e n c e o f an O-linked oligosaccharide moiety. A
positive staining was detected w h e n this protein was
treated with biotinylated jacalin, but not with c o n c a valin A suggesting the a b s e n c e o f an N-linked oligosaccharide moiety (data not s h o w n ) .
DISCUSSION
P
reviously, an 18-20 kDa protein was detected in
stool eluates from infected h u m a n and calves
with C. parvum,
but not in non infected control
samples (El-Shewy et al., 1 9 9 4 ) . In this report the
origin and nature o f this protein was elucidated.
T h e invasive sporozoites o f the apicomplexan parasites
possess secretory organelles located at the a p e x and
pellicle ( B l a c k m a n & Bannister, 2 0 0 1 ) . Among these
organelles, C. parvum
sporozoites reported to have
only o n e rhoptry (Tetley et ai, 1998). However, herein
w e demonstrated two rhoptries with many narrow
delicate ducts during TEM examination o f the C. parvum sporozoites.
Our findings with respect to the immunolocalization
of the 18-20 kDa protein included an anterior e n d protein extrusion, as detected on s o m e excysted sporozoites, while others were coated with this protein. This
labelling pattern could suggest a secretory nature of
the 18-20 kDa protein. It may b e p r o d u c e d internally
b y the secretory organelles and then translocated over
the surface or extruded via pellicle structures and
rhoptries ducts. A similar finding has b e e n reported in
Plasmodium
sporozoites, w h e r e an anterior trail of cir-
cumsporozoite protein was detected and believed to
b e synthesized internally, stored and released outside
b y appropriate host signals (Stewart & Vandenberg,
1991).
228
T h e detection o f completely electron-lucent secretory
organelles only in excysted and released sporozoites
after being electron-dense in the non excysted form
may support the secretory role o f these organelles. A
similar p h e n o m e n o n has b e e n o b s e r v e d in
Isospora,
Eimeria,
and in Plasmodium
merozoites (Bannister et
al, 1 9 7 9 ) .
SDS-PAGE analysis of sporulated C. parvum
sporozoites revealed more than 50 proteins o f which at least
40 a p p e a r e d to b e o f sporozoites origin (Tilly et al,
1991; Reperant et al., 1 9 9 2 ) . However, it w a s difficult
to evaluate the functional significance o f these proteins
in the d e v e l o p m e n t o f the sporozoites. With TEM, the
detected pattern was significantly related to the state
o f functional activity o f the sporozoites, as the non
excysted sporozoites did not s h o w any labeling. H o w e ver excysted, but not released sporozoites d e m o n s trated minimal labelling, finally, the typical gold labelling was o b s e r v e d w h e n sporozoites w e r e completely
released after excystation.
Despite the effect o f excystation o n the release o f the
18-20 kDa protein w e do not believe that it plays a
role in the e x c y s t a t i o n p r o c e s s . No labelling w a s
detected in excysted not released sporozoites. More
likely the detected 18-20 kDa protein has the s a m e
functions as those secreted in the a p i c o m p l e x a n parasites. In this respect, similarities in structures and functions of the apical proteins within apicomplexan genera
have b e e n d o c u m e n t e d (Grellier et al., 1994; Sam-Yellowe et al., 1988; Hehl et al., 2 0 0 0 ) . T h e s e proteins
appear to b e generally responsible for the selective
attachment to the host cells, formation o f the parasit o p h o r o u s v a c u o l e , and t h e s p o r o z o i t e s motility
( B l a c k m a n & Bannister, 2 0 0 1 ) . T h e distribution of 1820 kDa protein over the w h o l e surface and in front o f
the excysted sporozoites, represent a feature probably
shared by several a p i c o m p l e x a n parasites. T h e y glide
over surfaces on a carpet o f protein secreted from their
apical ends and distributed over their b o d i e s (Nus-
s e n z w e i g & Menard, 2000; Sultan et al., 2 0 0 1 ) . Accor-
E L - S H E W Y K.,
act as a lubricant or substrate for continuous gliding
motility o f the C. parvum
sporozoites.
T h e glycosylated nature detected by the affinity staihas b e e n reported to
to 9 0 0 kDa (Tilly et al.,
1 9 9 1 ; Barnes et al,
G R E L L I E R P.,
al.,
1999).
HEHL
Cryptosporidum
taken in our laboratory. O u r long aim is to d e v e l o p a
parasite.
LEKUTIS C.
G R I G G S M.E..
R.T. & S E K L A L. Purification of Cryptosporidium oocysts
and sporozoites by cesium chloride and percoll gradients.
American Journal of Tropical Medicine and Hygiene, 1985,
36. 5050-508.
W
LANGER
U . K . Cleavage of structural proteins during assembly
of the head of bacteriophage. Nature, 1970, 227(T4). 680685.
e thank Dr W. W e n m a n . Dr R. Kilani, and
R.C. &. R I G G S M.W. Cryptosporidium parvum apical
complex glycoprotein CSL contains a sporozoite ligand for
intestinal epithelial cells. Injection Immunity 1999. 6 7 ( 1 0 ) ,
5282-5291.
Diseases, and Medical Microbiology Univer-
sity o f Alberta, Alberta, Canada) for their g e n e r o u s
financial and technical support for a part o f this work.
F., M C C O L E D., E C K M A N N L. &
genesis of Cryptosporidium
paivum
and Infections 1999, 2, 141-148.
LAURENT
A R M B R U S T E R B.L.,
C A R L E M A L A M E.,
C H I I O V E T T I R.,
GARAVITO
K. & J O I N E R K.A. The parasitophorous vacuole
membrane surrounding Plasmodium and Toxoplasma: an
unusual compartment in infected cells. Journal of Cell
Sciences. 1998. 111, 1467-1475.
R.M.,
J.A., K E L L E N B E R G E R E. & V I L L I G E R W. Specimen preparation for electron microscopy using low temperature
embedding resins. Journal of Microscopy, 1982, 126, 7785.
BONNIN
A.,
HUANG J-X.
G O U S S E T L., W U . J . , G U T
J.,
A novel
multidomain mucin-like glycoprotein of
Cryptosporidium
parvum mediates invasion. Molecular Biochemical
Parasitology,
1998. 96, 93-110.
P.,
DOYLE
DUBERMEETZ
J.F.,
WARD
H. &
PETERSEN
C.
M . J . & B A N N I S T E R L . H . Apical organelles of Apicomplexa: biology and isolation by subcellular fractionation. Molecular Biochemical Parasitolology, 2001, 117, 1125.
BLACKMAN
BANNISTER
L.H.,
BUTCHER
G.A.,
DENNIS E.D.
&
MICHELL
G.H.
Structure and invasive behavior of Plasmodium
knowlesii
merozoites in vitro. Parasitology.
1979. 77, 483-491.
CURRENT
W.L. &
tosporidium
M.F. Pathoinfection. Microbes
KAGNOFF
LINGELBACH
HOBOT
B A R N E S D.A..
DUBREMETZ
KILANI
LAEMMLI
REFERENCES
B R A D L E Y P.J.,
ORTEGA-BARRIA
ACKNOWLEDGEMENTS
Mr. R. S h e r b u r n e (Division o f Infectious
& SCHREVEL J .
E. & C L A D A. Electroelution of fixed and stained
remembrance proteins from preparative sodium dodecylsulfate polyacrylamide gels into a membrane trap. Analytical Biochemistry,
1986, 154, 583-589.
characterization o f this antigen protein is being under-
inhibition o f the Cryptosporidium
CARCY B.
JACOBS
vaccine d e v e l o p m e n t . Molecular cloning and further
protective e l e m e n t for neutralization and/or invasion
V A L E N T I N A.,
E. & B O O T H R O Y D J.C. Toxoplasma gondii
homologue of Plasmodia
apical membrane antigen 1 is
involved in invasion of host cells. Infection
Immunity.
2000, 68. 7078-7086.
glycosylated e x o a n t i g e n with an
enterotoxic activity that could b e potential target for
A.B.,
J.F..
T h e data presented in this study could throw lights on
a
P R E C I G O U T E.,
Characterization of a new 60 kDa apical protein of Plasmodium falciparum
merozoites expressed in late schizogony. Biology of the cell, 1994, 82, 129-138.
20 kDa protein, an enterotoxic activity was remarkably
heat labile and chloride d e p e n d e n t (El-Shewy et
WENMAN
R. & U N G A R B.L.P. Cryptosporidium and crypotosporidiosis. Microbiology Review, 1986, 50. 458.
o b s e r v e d on using Ussing c h a m b e r . T h e enterotoxic
effect o f this protein was time and d o s e dependent,
L. &
FAYER
1998). In
a previous trial for further characterization o f this 18-
MAKHLOUF
K., H U S S I E N A.M. & E L - M A G R A B I D.F. An enterotoxic
activity of 18-20 kDa Cryptosporidium
secretory coproantigen (18-20 kDa CCA). Journal of Egyptian Society of Parasitology,
1999, 2 9 (1), 281-289.
possess glycoprotein located on the surface o f its s p o rozoites and having molecular weights range from 15
H E G A Z I M.M..
EL-SHEWY
ning denotes the potential immunogenicity o f this protein. In other studies, C. parvum
K I L A N I R.T.,
W.M. Identification of low molecular weight coproantigen
of Cryptosporidium parvum. Journal of Infectious Diseases.
1994, 169. 460-463.
dingly w e anticipated that the detected protein might
H A Y N E S T . B . Complete development of Crypin cell culture. Science, 1984, 224, 603-605.
MA
P. & S O A V E R. Three steps stool examination of cryptosporidiosis in ten homosexual men with protracted watery
diarrhea. Journal of infectious Diseases, 1983, 147, 824-828.
W.R., S C H E L L W.L., B L A I R K.A., Anniss D.G., P E T E R S O N
D.E. & H O X I E N . J . Massive outbreak of
Ciyptosporidium
infection in Milwaukee, Wisconsin: recurrence of illness
and risk of secondary transmission. Clinical
Infectious
Diseases, 1995, 21, 57-62.
MACKENZIE
V . & M E N A R D R. Analysis of a malaria sporozoite
protein family required for gliding motility and cell invasion. Trends in Microbiology, 2000, 8, 94-96.
NUSSENZVCEIG
M.E. Rhoptry organelles of Apicomplexan parasites.
Parasitology Today. 1992. 8. 28-32.
PERKINS
P R E I S E R P.,
K A V I R A T N E M.,
K H A N S.,
B A N N I S T E R L.H.
& JARRA
W.
The apical organelles of malaria merozoites: host cell selection, invasion, host immunity and immune evasion. Microbes
and Infection, 2000. 2, 1461-1477.
229
J.. NACIRI M., C H A R D E S T . & B O I T D.T. Immunological characterization of a 17-kDa antigen from Cryptosporidium parvum recognized early by mucosal IgA antibodies. FEMS. Microbiology Letter 1992, 99, 7-14.
REPÉRANT
R. & H O L D E N D.W. Protein blotting: detection of
proteins with biotin-conjugated and enzymes probes.
Annuals of Biochemistry,
1985, 144, 118-127.
ROHRIXGER
R O T H J.,
BENDAYAN
M.,
CARLEMALAM
E.,
WERNER
V.
&
GARA-
\ I T O M. Enhancement of structural preservation and immunocytochemical tissue. Journal of Histochemistry and Cytochemistry, 1981, 29, 663-671.
T . Y . , S H I O H . & P E R K I N S M.E. Secretion of Plasmodium falciparum
rhophty protein into plasma membrane of host erythrocytes. Journal of Cell Biology 1988,
106, 1507-1513.
SAM-YELLOWE
J.D. Inhibition of penetration-enhancing factor
of Toxoplasma gondii by monoclonal antibodies specific
for rhoptries. Infection Immunity, 1986, 55, 760-764.
SCHWARTZMAN
P.G. & D O D D M . C . Self-aggregation of bovine skin proteodermatan sulphate promoted by removal of three relinked oligosaccharides. Connective Tissues Research, 1990,
24, 225-236.
SCOTT
J . M . & V A N D E R B E R G P.J. Malaria sporozoites release
circumsporozoite protein from their apical end and translocated it along their surface. Journal of Protozoology, 1991,
38 (4), 411-121.
STEWART
SULTAN
A.A.,
THATHY
V.,
DE-KONING-WARD
T.F.
&
NUSSENZ-
Complementation of Plasmodium
berghei TRAP
knockout parasite using human dihydrofolate gene as a
selectable marker. Molecular Biochemical
Parasitology.
2001, 113. 45-53.
WEIG
V.
L., B R O W N S.M., M C D O N A L D S V . & C O O M B S G . H . Ultrastructure analysis of the sporozoite of
Ciyptosporidium
parvum. Microbiology.
1998. 144. 3249-3255.
TETLEY
T I L L Y M.,
U P T O N S.J.,
F A Y E R R.,
BARTA JR.,
C H R I S P C.E.
&
FREED
P.S. Identification of a 15-kilodalton surface glycoprotein
on sporozoites of Cryptosporidium parvum. Injection Immunity, 1991, 59. 1002-1007.
H . T . , S T A E H L I N T . & G O R D O N J. Electrophoretic transfer
of protein from polyacrylamide gel to nitrocellulose sheets:
procedure and some application. Processing Natural Academy of Sciences USA, 1979, 76, 4350-4354.
TOWBIN
W.M., K Y L E J.E.. S P E E R C.A. & B U R G E S S D.E. Inhibition of penetration of cultured cells by Eimeria bovis sporozoites by monoclonal immunoglobulin G antibodies
against the parasite surface protein P20. Infection
Immunity, 1986. 56. 2538-2543.
WHITMIRE
Reçu le 1 avril 2003
Accepté le 9 décembre 2003
er
230