GASTROENTEROLOGY 1986;90:583-94
Cryptosporidium: Cellular Localization,
Structural Analysis of Absorptive Cell­
Parasite Membrane-Membrane
Interactions in Guinea Pigs, and
Suggestion of Protozoan Transport
by M Cells
MANUEL A. MARCIAL and JAMES 1. MADARA
Department of Pathology (Gastrointestinal Pathology) , Brigham and Women's HospitaL Harvard
Medical School. and the Harvard Digestive Diseases Center, Boston, Massachusetts
In ilea of spontaneously infested guinea pigs, we
examined the interface between the plasma mem­
branes of cryptosporidia and absorptive cells using
thin section and freeze fracture techniques. Initially,
cryptosporidia invaginate microvilli, and the result­
ing redundant folds of membrane envelop the pro­
tozoan, thereby internalizing it in a membrane sac of
host cell origin . Subsequently, a pentalaminar mem­
brane fusion site develops at the base of the proto­
zoan between the parasite's outer plasma membrane
and the internalized host membrane. The membrane
domains isolated by this fusion site are then modi­
fied: the host membrane disintegrates, and the iso­
lated parasite membrane, which now directly con­
tacts absorptive cell cytoplasm, becomes amplified.
While cryptosporidia are restricted to the apex of
absorptive cells, they may be found deep within the
cytoplasm of M cells overlying Peyer's patches.
Moreover, both intact and partially digested cryp­
tosporidial organisms associate with macrophages
subjacent to such M cells. These findings define the
intracellular localization of cryptosporidia and sug­
gest that cryptosporidial antigens may be sampled
by intestinal lymphoid cells at sites underlying M
cells .
Received June 21, 1985 . Accepted September 3, 1985.
Address requests for reprints to: James L. Madara, M.D., Depart­
ment of Pathology, Brigham and Women's Hospital. 75 Francis
Street, Boston, Massachusetts 02115.
This work was s\,lpported by National Institutes of Health grant
AM 35932 and National Research Service Individual Fellowship
AM 07012. Dr. Madara is supported, in part. by the American
Gastroenterological AssociationlRoss Research Scholar Award.
The authors thank Susan Carlson for expert technical assistance
and Mary LoGiudice for preparation of the manuscript.
© 1986 by the American Gastroenterological Association
0016-5085/86/$3.50
Organisms of the genus Cryptosporidium (phylum
Apicomplexa, class Sporozoea, subclass Coccidia)
(1) produce a parasitic disease that involves the
gastrointestinal, biliary, and/or respiratory epithe­
lium of many animal species, including nonhuman
mammals, reptiles, birds (2-4), and more recently,
humans (5,6). Early reports of this disease in humans
described an associated, protracted, watery diarrhea
occurring in immunosuppressed patients, many of
whom had acquired immunodeficiency syndrome
(AIDS) (7,8). Recent epidemiologic studies utilizing
techniques that identify Cryptosporidium oocytes in
stool specimens have shown that cryptosporidiosis
may also present as an acute, self-limited diarrheal
disease in immunocompetent individuals and may
account for 1%-10% of diarrheal disease among the
population at large (9-11).
Tyzzer (12), who first described this protozoan,
defined it as an extracellular parasite (13), a view
that was not challenged until 1966 (14) . Subse­
quently, several transmission and scanning electron
microscopic studies have approached the issues of
the anatomy of the host cell-parasite relationship
and of the cellular localization of the protozoan
(15-18). In general, such studies lacked detailed,
high-resolution examination of parasite and host cell
membranes, and thus, not surprisingly, they have
come to divergent and discrepant conclusions
(14,16) as to the intracellular or extracellular local­
ization of the protozoan.
Using thin section and freeze fracture techniques,
we performed detailed ultrastructural analysis of the
small intestinal epithelium of guinea pigs spontane­
ously infected by Cryptosporidium. The anatomy of
host and parasite membranes was carefully analyzed
584
MARCIAL AND MADARA and, using the distinctive life cycle forms of the
parasite, the sequential interactions between mem­
branes of the two cell types were assessed.
Lastly, as the membranous epithelial (M) cells (19)
overlying the gut-associated lymphoid tissue are
capable of transporting both viral and bacterial orga­
nisms (20,21), we performed ultrastructural studies
of Peyer's patch epithelium to see if we could iden­
tify transport of this parasite by the M cell.
Materials and Methods
Small intestinal mucosa from three adult male
Hartley strain guinea pigs, Cavia porcellus (Elm Hill
Farms, Chelmsford, Mass.) weighing 400-600 g, were
studied. The spontaneous cryptosporidial infection in
these guinea pigs was serendipitously discovered in the
course of harvesting control tissues for other experiments.
In our series of > 300 guinea pigs used for experimental
studies, these have been the only animals recognized as
GASTROENTEROLOGY Vol. 90, No.3
being infected by cryptosporidia. The guinea pigs had
been fasted overnight but were allowed to drink water ad
libitum. Under urethane anesthesia, segments of ileum,
including Peyer's patches, were excised, opened longitu­
dinally, and fixed by submerging them in a 4°C solution of
2% formaldehyde and 2.5% glutaraldehyde in 0.1M so­
dium cacodylate buffer at pH 7.4 for 2 h (22). Subse­
quently, tissues for light and electron microscopy were
postfixed in 1% osmium tetroxide, dehydrated in graded
concentrations of ethanol, and embedded in Epon. Ori­
ented 1-p,ID-thick sections were obtained and stained for
light microscopy with toluidine blue in 0.2M phosphate
buffer pH 7.0. Epon blocks were then trimmed and silver­
interference-color thin sections were cut with a diamond
knife. The sections were mounted on No. 200 hexagonal
mesh copper grids and stained with uranyl acetate and
lead citrate.
In addition, non-Peyer's patch mucosal samples were
prepared for freeze fracture. The tissues were fixed as
above, washed three times in sodium cacodylate buffer,
embedded in a 3% agar solution, and chopped into
125-p,ID slices with a Smith-Farquhar tissue chopper
B
Figure 1. A. Schematic diagram of a Cryptosporidium outlining the results of our structural findings for the early trophozoite phase. For
descriptive purposes we have divided the organism into two general areas-that which indents the terminal web (B) and that
which is above the terminal web (A) . In the area above the terminal web five unit membranes are identified , labeled 1 through
5. These represent: 1, envelope's outer membrane; 2, envelope's inner membrane; 3, parasite's plasma membrane; 4 and 5, an
inner pair of membranes intrinsic to the parasite. Together, membranes 3, 4, and 5 correspond to what others have termed the
pellicle of the parasite. With the exception of the envelope's outer membrane (membrane 1). all membranes are also present
below the terminal web at this early trophozoite phase. In addition, the area below the terminal web has a dense band
subjacent to the parasite-absorptive cell interface. This dense band represents an absorptive cell cytoskeletal specialization
(see Results). B. Electron micrograph of an early trophozoite showing the various membranes in both general areas of the
parasite. This trophozoite is identified as an early form by the presence of rhoptries (R). the absence of endoplasmic reticulum,
the nuclear size (NJ, and by its elliptical rather than oval shape. (This last feature cannot be fully appreciated in this
micrograph because it contains only a segment of the parasite.) The electron-lucent appearance of the rhoptries suggests that
they have released their contents . The envelope and parasite membranes have been labeled to correspond to those in the
accompanying schematic diagram. Note that, focally, the parasite's plasma membrane is becoming closely apposed to
membrane 2 (arrowhead). DB represents dense band. Uranyl acetate and lead citrate. Approximately x 96,000.
March 1986
ANATOMY OF INTESTINAL CRYPTOSPORJDIOSIS
585
Figure 2. A. Electron micrograph of the multiple membrane fracture planes of a Cryptosporidium (arrowheads) that is attached to an
underlying absorptive cell (AC). Membranes are labeled as in the schematic diagram shown in Figure 1: 1, envelope's outer
membrane; 2, envelope's inner membrane; 3, 4, and 5, parasite's three-membrane pellicle. Note that the fracture face of the
envelope's outer membrane is continuous over the superior pole of the protozoan. Approximately x 27,000. Inset:
magnification of the envelope's outer membrane P-fractore face. Note that this fracture face has an intramembrane particle
density similar to that of the adjacent absorptive cell microvillus membrane P faces (MV). Approximately x57,QOO. B. Highly
magnified electron micrograph of the replicated interface between a parasite (P) and an absorptive cell (AC) with its associated
microvilli (MV) . As highlighted by the small arrowheads, the envelope's outer membrane , labeled 1, is continuous with the
microvillus membrane. The envelope's inner membrane (membrane 2), which presumably represents internalized microvillus
membrane, separates a fractured membrane of the parasite from direct contact with host cell cytoplasm. Note that the host cell
cytoplasm is continuous with the envelope cytoplasm (large arrowhead). The site of the dense band seen in thin section (see
Figure 4) is indicated by the arrow, and at this site no membrane fracture face is revealed. Approximately x 105,000. In both
photomicrographs the direction of platinum shadowing is roughly bottom to top.
,
(Sorvall, Newton, Conn.). After equilibrating for 1 h in a
solution of 25% glycerol in O.lM sodium cacodylate buffer,
each tissue slice was mounted between two gold disks,
rapidly frozen in the liquid phase of partially solidified
Freon 22, and stored in liquid nitrogen. Tissues were
fractured at a stage temperature of -110°C and a vacuum
pressure of <6 X 10- 7 mmHg in a Balzers 300 freeze-etch
device (Balzers, Hudson, N.H.) equipped with a double
replica attachment. The fractured tissue was replicated
with platinum and carbon without etching, cleaned in
commercial bleach, rinsed in distilled water, and mounted
on Formvar-coated (Ernest F. Fullam, Inc., Schenectady,
N.Y.) No. 200 hexagonal mesh copper grids. Both freeze
fracture replicas and silver thin sections were examined
and photographed in a Philips 201 electron microscope
(Philips Electronic Instruments, Inc., Mahwah, N.J.) at 60
kV . A total of 400 electron micrographs (206 of thin
sections and 194 of freeze fracture replicas) were analyzed.
Because in thin sections the various stages of the life cycle
of Cryptosporidium, as well as the various maturational
phases of trophozoites, are ultrastructurally distinctive
(15), as briefly outlined below, we were able to draw
586
GASTROENTEROLOGY Vol. 90, No. 3
MARCIAL AND MADARA
I'- j
Figure 3. Electron micrograph of a maturing trophozoite (P) that has lost its rhoptries and is beginning to develop endoplasmic
reticulum. This trophozoite , as are all trophozoites shown, is attached to an absorptive cell (AC). Note that the envelope
cytoplasm contains parallel arrays of microfilaments seen best when tangentially sectioned (arrowheads). The inset shows an
envelope at high magnification that consists of an electron-dense cytoplasmic core sandwiched between two membranes (1
and 2). Underlying the trophozoite-absorptive cell interface is a dense band (DB) , but no microvillus rootlets are seen at this
site. Uranyl acetate and lead citrate. Approximately X48,000. Inset: Approximately x 204 ,000.
conclusions from our material concerning the sequence of
morphologic events associated with attachment of this
parasite to absorptive cells. Early trophozoite forms are
identified by their elliptical to ovoid shapes, absence of
rough endoplasmic reticulum , and incomplete dedif­
ferentiation of the apical complex (15). The apical com­
plex, which ultrastructurally defines protozoans as mem­
bers of the phylum Apicomplexa (23). consists of several
specialized organelles that are believed to aid in parasite
penetration into host cells . These include the rhoptries
(electron-dense club-shaped organelles), the polar ring (an
osmiophilic thickening formed by the inner membranous
layer of the pellicle). and the micronemes (small,
osmiophilic convoluted structures) (23) . These organelles,
which are identifiable in early trophozoites, disappear as
the trophozoite matures (15) . Ultrastructural evidence of
trophozoite maturation also includes rounding of the par­
asite into a spherical body, enlargement of the nucleus and
nucleoli, the development of the rough endoplasmic
reticulum cysternal complex, and the development of a
zone with interdigitated membranous folds at the host­
parasite interface (15). The nucleus of the fully matured
trophozoite finally undergoes a series of divisions fol­
lowed by cytokinesis, and schizonts are formed.
March 1986 ANATOMY OF INTESTINAL CRYPTOSPORIDIOSIS
587
Figure 4. Electron micrograph of the interface between an absorptive cell (AC) and a maturing trophozoite (P). Toward the base of the
parasite, as shown by the arrowheads, the parasite plasma membrane (membrane 3 in Figure 1 schematic) and the internalized
microvillus membrane (membrane 2 in Figure 1 schematic) form a discrete pentalaminar membrane fusion site. Two
membrane domains are isolated by this fusion site, a disk of membrane 3 (parasite origin) and a disk of membrane 2 (host
origin). Dense band can be seen below membrane 2. Uranyl acetate and lead citrate. Approximately X167,OOO.
Results
Ileal Villus Epithelium
Light microscopy. The architecture of the
small intestinal mucosa of infested animals was
altered, showing a 2:1 villus height to crypt depth
ratio. (Normally, a 3:1 to 4:1 ratio is present.) A
sparse infiltrate of polymorphonuclear leukocytes
was present in the lamina propria and the epithe­
lium. These findings were interpreted as evidence of
a mild enteritis.
Organisms, measuring from 2 to 4 /-tm, were asso­
ciated with the villus absorptive cell brush border.
Transmission electron microscopy. With the
exception of oocysts and free zoites (sporozoite or
merozoite), multiple examples of the various
Cryptosporidium life cycle forms (15) were identi­
fied by thin section. We predominantly observed
various maturational forms of trophozoites, how­
ever, and have concentrated on detailing sequential
anatomic alterations accompanying this phase of the
life cycle. In freeze fracture replicas, identification of
the specific life cycle stages of individual parasites
using the above, previously described, thin section
criteria was not always possible. As outlined below,
however, thin section examination did show struc­
tural rearrangement of specific membrane domains
during trophozoite maturation. Thus, by using these
characteristic alterations in membrane surfaces to
identify various maturational forms, we were usu­
ally able to distinguish these forms in replicas as
well as in thin sections. Because of the number of
membranes dealt with, we will refer to the schematic
diagram in Figure 1 in the following descriptions of
our electron microscopic images. For descriptive
purposes, we divide the protozoan into two general
areas-that which indents the terminal web, and that
above the terminal web where the parasite lies
within the microvillus border (Figure 1).
We will first describe the appearance of mem­
branes surrounding early or immature trophozoites.
In the area above the terminal web (Figure 1). five
unit membranes were associated with these early
trophozoite forms. The outermost membrane (la­
beled 1 in Figure 1) was continuous over the supe­
rior pole of the parasite and occasionally could be
seen to be covered by glycocalyx. Furthermore, this
outer membrane was in continuity with the absorp­
tive cell apical membrane (Figure IB). The continu­
588
MARCIAL AND MADARA GASTROENTEROLOGY Vol. 90, No.3
Figure 5. High power electron micrograph of the interface of an absorptive cell (AC) and a mature trophozoite (P) identified as such by
the structural characteristics of the cytoplasm (absent rhoptries, large size, presence of endoplasmic reticulum). Although the
pentalaminar fusion site is still present (arrow), the isolated disk of host membrane (membrane 2 in Figure 1 schematic),
previously located at the site marked by the arrowheads, has been lost. The disk of parasite plasma membrane isolated by the
fusion site has undergone extensive infolding and amplification and now has direct access to the host cell cytoplasm, although
host cell cytoskeletal elements do not penetrate into the interdigitations produced by these folds . Lastly, the two inner
membranes of the pellicle (membranes 4 and 5 in Figure 2 schematic) have been lost. E, envelope; MV, microvilli. Uranyl
acetate and lead citrate. Approximately x 105,000.
ity of this outer membrane, both over the parasite
(Figure 2A) and with the apical membrane of adja­
cent microvilli (Figure 2B), was confirmed in freeze
fracture replicas. Also, as seen in the inset of Figure
2A, the P fracture face of this outer membrane had a
high intramembrane particle density similar to that
seen on the P faces of absorptive cell microvilli. The
cytoplasm lying directly under this outer membrane
was of the same electron density as the microvillus
core cytoplasm and contained parallel arrays of
microfilaments (Figure 3).
Moreover, in fractured parasites, the host cell
cytoplasm was shown to be continuous with the
cytoplasm underlying this outermost membrane sur­
. rounding the parasite (Figure 2B). The second mem­
brane (labeled 2 in Figures 1 and 3) separated this
host cell cytoplasmic extension, which enveloped
the parasite, from a space that surrounded the para­
site. Thus, the two outermost membranes, which we
will refer to as the parasite's envelope membranes,
are of host cell origin and contain between them a
narrow rim of host cell cytoplasm. The inner enve­
lope membrane represents internalized microvillus
membrane that has been invaginated by the proto­
zoan during the process of attachment. Within the
zone of the brush border, the fracture planes of these
envelope membranes revealed an undulating pattern
that, on cross-fractured planes, seemed to corre­
March 1986 ANATOMY Of INTESTINAL CRYPTOSPORIDIOSIS
589
Figure 6. Electron micrograph of fractured membrane planes at the absorptive cell-parasite interface. A portion of the host membrane,
labeled 2, which presumably corresponds to the disk of this membrane isolated by the pentalaminar fusion site, has a ruffled
surface appearance similar to that seen in detergent-extracted membranes. Thus this image may correspond to the early phase
of lysis of this membrane disk, which follows the pentalaminar fusion shown in figure 5 (arrowheads). The parasite's outer
membrane is labeled P. AG, absorptive cell. Approximately x 83,000.
spond to indentations produced by surrounding mi­
crovilli.
The inner three unit membranes (Figure 1). which
intimately associated with the parasite and were
separated from the envelope membranes by the
aforementioned space, had no continuity with host
cell membranes and thus represented membranes
intrinsic to the parasites. These three membranes
were also identified in individual merozoites within
schizonts and correspond to the three-membrane
pellicle that characterizes zoites of parasites belong­
ing to the class Sporozoaea, subclass Coccidia
(23,24). The pellicle or covering of the parasitic
surface consists of an outer membrane or plasma
membrane and an inner pair of membranes (Figure
1B) that are closely opposed to one another. By
freeze fracture, the protozoan's plasma membrane
(labeled 3 in Figure 1) revealed "P" faces with low
intramembrane particle areal densities, as did the
most exterior of the inner duet of membranes, la­
beled 4 in Figure 1, whereas the remaining mem­
brane, labeled 5 in Figure 1, had an intermediate
number of large intramembrane particles (Figure
2A).
In areas where the early trophozoites indented the
terminal web, four, instead of five, unit membranes
were identified: the inner envelope membrane, and
the three membranes of the so-called parasite
pellicle (Figure 1). A narrow space separated the
inner envelope membrane from the parasite's plasma
membrane. Thus, the inner envelope membrane sep­
arates, at this stage of the life cycle, the parasite from
direct contact with host cell cytoplasm (Figure 2B).
Within the terminal web, at sites near the para­
site's interface with the absorptive cell, there was an
electron-dense structure, which has previously been
referred to as the "dense band" (15) (Figures 3 and
4). An ultrastructural periodicity was sometimes
seen, but no unit membrane structure was ever
demonstrated on thin section and no membrane
fracture plane was visualized in this area in freeze
fracture replicas (Figure 2B) . Thus, the dense band
represents a host cell cytoplasmic specialization and
not the true interface between parasite and host cell.
Microvillus rootlets were absent in the terminal web
region under the parasite (Figure 3).
Analysis of trophozoites at different phases of
maturation, as identified by changes in organelle
composition, revealed that consistent, maturation­
dependent modifications in membrane structure oc­
curred. First, the inner pair of membranes of the
parasite pellicle regressed, leaving the outer or
plasma membrane as the only membrane over most
of the lateral and lower surfaces of the protozoan
(Figure 5). Second, early in maturation, the inner
envelope membrane of host origin and the parasite's
plasma membrane approximated one another at a
level just below that where the parasite indented
into the terminal web (Figure 1B). Subsequently,
these two membranes joined to form a pentalaminar
membrane fusion site (Figure 4). These fusion sites
isolate two distinct membrane domains, one of par­
asite plasma membrane and the other of internalized
590 MARCIAL AND MADARA GASTROENTEROLOGY Vol. 90, No.3
~ f;" ,
I'
.'
Figure 7. After maturation, a trophozoite undergoes divisions to produce mUltiple merozoites (MZ). as seen in this electron micrograph
of a first-generation schizont (P). Note the three-membrane pellicle around each individual merozoite. These merozoites will
eventually be released and will attach to other absorptive cells. At this stage cytoplasmic structures such as micronemes (MNJ,
which also characterize immature trophozoites, reappear in merozoites. The membrane organization at the absorptive cell
(AC)-parasite interface still consists of an amplified parasite plasma membrane that has direct contact with host cell cytoplasm
and rests above the dense band (DB). Uranyl acetate and lead citrate. Approximately x37,OOO.
microvillus membrane (inner envelope membrane)
(Figure 4). We were not able to obtain good quality
replicas of this fusion zone at this particular stage of
development and thus are unable to comment on its
freeze fracture appearance. The internalized
microvillus membrane domain isolated by the
pentalaminar membrane fusion site underwent pro­
gressive dissolution (Figure 5). In one fortunate
fracture plane where this membrane was visualized
while undergoing dissolution, it had, at its base,
structural characteristics similar to those observed in
replicas of detergent-extracted membranes (Figure
6). This process of host membrane dissolution led to
the establishment of a direct contact between the
domain of parasite plasma membrane isolated by the
fusion sites and the host cell cytoplasm. Subse­
quently, again as suggested by analysis of different
phases of maturation, this latter parasite membrane
domain was amplified to form a complex system of
membrane folds-a structure which has previously
been referred to as the "feeder organelle" (2,16)
(Figure 5). This structure was seen in replicas as
multiple membrane facets.
The structural organization of the membranes at
the parasite-host cell interface during the schizont or
gamete stage of the life cycle was comparable to that
described above for the mature trophozoite (Figure
7). However, even though most of the mature tropho­
zoite pellicle was composed of a single unit mem­
brane, the individual merozoites within a schizont
had the characteristic three-membrane pellicle as
mentioned earlier (Figure 7) .
Mucosa Overlying Peyer's Patches
Light microscopy . The follicle-associated
dome epithelium was not structurally disturbed.
Organisms were rarely identified in the brush border
March 1986 ANATOMY OF INTESTINAL CRYPTOSPORIDIOSIS
591
of epithelial cells that overlie the domes of Peyer's
patches. Presumptive M cells were identified by
their characteristic structural appearance, which in­
cludes attenuated brush borders, clearer cytoplasm
relative to absorptive cells, and an association with
aggregates of intrusive interepithelial lymphoid
cells.
Transmission electron microscopy. Cryp­
tosporidia were identified associated with follicular
dome absorptive cells and with both mature (25) and
immature M cells (25). Ultrastructural criteria used
for the identification of M cells were those outlined
in detail in the literature, which include microvilli
that are shorter, wider, and more irregular than those
of absorptive cells; terminal webs that are less well
defined than in absorptive cells; increased cytoplas­
mic vesicles; and the association with intrusive
phagocytic lymphoid cells (19,25,26). The associa­
tion of cryptosporidial organisms with dome absorp­
tive cells was identical to that described above for
villus absorptive cells. In striking contrast, these
parasites were identified deep within the cytoplasm
of both immature and mature (Figure 8) M cells.
All the organisms seen within M cells were- either
in the merozoite or trophozoite life cycle stage. The
structural modifications of membranes that were
described as occurring at the parasite-absorptive cell
interface were not seen in M cells, although the
sample we were able to examine in our material was
limited. Additionally, in no instance were dense
bands noted adjacent to internalized parasites. Sur­
face attachment of parasites to M cells was also not
observed in our sample. Both intact and partially
digested cryptosporidial organisms were seen in
association with macrophages underlying such M
cells (Figure 8B).
Discussion
Using electron microscopy of thin sections
and freeze fracture replicas we demonstrate defini­
~---------------------------------
Figure 8. A. Electron micrograph of a mature M cell (MC) con­
taining multiple internalized parasites (P). At higher
magnification, cytoplasmic micronemelike organelles
were identified in the organism closest to the lumen.
These organisms, based on this finding and their size,
probably represent merozoites. Uranyl acetate and lead
citrate. B. Electron micrograph of follicular dome epi­
thelium revealing a mature M cell (MC). Cryptosporid­
ial organisms (P) are seen to be associated with macro­
phages subjacent to the M cell. Organisms were never
seen in such a location in villus epithelium, even
though our sample from villus epithelium was exten­
sive compared to our relatively small sample of follic­
ular dome epithelium. Uranyl acetate and lead citrate.
L, lymphoid cells. Both A and B are approximately
x 6000.
592
GASTROENTEROLOGY Vol. 90 , No.3
MARCIAL AND MADARA ®
©
®
@
Figure 9. Schematic diagrams depicting the progression of cryptosporidial trophozoite-absorptive cell interactions based on our
observations of various phases of trophozoites. As cryptosporidia invaginate absorptive cell microvilli (AJ, the resulting
redundant folds of apical membrane envelop the organism (B). This results in the intracellular localization of the organism
within a sac of internalized microvillus membrane. The plasma membrane of the Cryptosporidium subsequently fuses toward
its base with this invaginated host membrane (B). The two membrane domains isolated by this process subsequently undergo
drastic alteration (C and D) . The host membrane dissolves (C), and the isolated parasite plasma membrane, which now directly
contacts host cell cytoplasm, becomes amplified (D) . During this progression, the inner two membranes of the parasite pellicle
(labeled 4 and 5) disappea.r, particularly toward the base of the parasite (B and C).
March 1986 tively the intracellular localization of cryptospori­
dial trophozoites. The specific sequence suggested
by our structural findings is outlined in Figure 9.
These organisms were shown to be enveloped by a
thin host cell-derived envelope. The host cell­
derived envelope most likely results from the para­
site invaginating the microvillus membranes during
early attachment. Such membrane invagination is
the method of host cell entry for other coccidia and
perhaps for all sporozoans (27-30). Our structural
observations suggest that the resulting redundant
folds of apical membrane subsequently surround the
protozoan and eventually fuse over the superior pole
of the organism. The inner membrane of the enve­
lope would thus represent an internalized sac of
microvillus membrane that initially separates the
parasite plasma membrane from direct contact with
the host cell cytoplasm. Our data provide evidence
that, at a specific anatomic domain, the surface of the
parasite plasma membrane subsequently achieves
intimate contact with the host cell cytoplasm. This
association appears to be achieved by a series of
remarkable structural events requiring modifications
at highly specific membrane domains on both host
cell and parasite.
After envelopment by the host cell, the plasma
membrane of the parasite and the internalized
microvill us membrane focally form a discrete
pentalaminar membrane fusion site, which may rep­
resent a variation of the "moving junction" seen as
Plasmodium morozoites indent host cells (30,31).
This fusion site isolates both a disklike host cell and
a disklike parasite membrane domain that subse­
quently undergo drastic alteration; the host mem­
brane dissolves, and the isolated parasite membrane,
which now directly contacts host cell cytoplasm,
eventually becomes amplified. This latter membrane
amplification has been referred to as the "feeder
organelle" (2,16)-a label that suggests, logically
considering our findings, that this is the site at which
nutrient uptake from the host cell cytoplasm occurs.
We also show that the dense bands underlying the
parasite attachment site are not unit membranes but
appear to be areas of modified host cell cytoskeleton.
The function of this band is unclear, but it might
serve to anchor the parasite to the host cell or,
alternatively, resist further invasion into the absorp­
tive cell cytoplasm. We have previously described
similar cytoskeletal specializations at the sites where
filamentous bacteria invaginate into the cytoplasm of
rodent ileal absorptive cells (32).
We found Cryptosporidium to have pellicles com­
posed of three membranes: a plasma membrane and
an underlying pair of membranes. These latter two
membranes were closely apposed to each other and,
in some developmental stages, were not discernible,
ANATOMY OF INTESTINAL CRYPTOSPORIDIOSIS
593
particularly at the base of the organism. Although
this three-membrane pellicle is characteristic of the
protozoans belonging to the subphylum Apicom­
plexa (23), especially in the motile stages (sporo­
zoites and merozoites), it had not been previously
described for Cryptosporidium. The inner mem­
brane complex of the pellicle is thought to originate
from the flattening of vesicular organelles, such as
endoplasmic reticulum, during schizogony. As men­
tioned above, there is partial disappearance of this
inner membrane complex with trophozoite matura­
tion-a process that also affects the specialized
organelles of the apical complex, and has been
described as occurring in other coccidia and plas­
modia (24,33).
Cryptosporidial organisms are the first example of
a parasite to be identified within the cytoplasm of M
cells. Although Giardia muris has been identified
within macrophages in Peyer's patch lymphoid fol­
licles, it was not clear if these organisms gained
access to these macro phages by way of transport
through M cells or by invasion between dome epi­
thelial cells (34,35). Membranous epithelial cells
previously have been shown to endocytose and
transport other microorganisms (including viruses
and bacteria) from the lumen to underlying
phagocytic lymphoid cells (20,21,32,34). This path
of entry presumably permits antigenic sampling by
the intestinal immune system and may represent an
essential component of mucosal immunity. Compe­
tence of the intestinal immune system and its ability
to respond to the stimulus presented via M-cell
endocytotic transport may explain why cryptospori­
diosis is a self-limited disease in immunocompetent
hosts.
The intracellular location of Cryptosporidium
might potentially explain the difficulties encoun­
tered in eradicating this organism in immunocom­
promised patients (4). We speculate that, as is the
case with other intracellular organisms (36), thera­
peutic agents that wil1 effectively eradicate cryp­
tosporidiosis have to be able to penetrate absorptive
cell plasma membranes. This suggestion should sen­
sitize investigators to the possibility that while the in
vitro culture system for Cryptosporidium allows
progress in the study of areas such as the protozoan's
life cycle (37,38), such a system may have limita­
tions as a screening method for agents that will
eradicate the organism under in vivo conditions.
References
1. Levine ND. Corliss )0. Cox FEG. el al. A newly revised
classification of the protozoa. ) Protozool 1980;27:37-58.
2. Tzipori S. Cryplosporidiosis in animals and humans. Micro­
bioi Rev 1983;47:84-96.
594
MARCIAL AND MADARA 3. Angus KW. Cryptosporidiosis in man, domestic animals and
birds: a review. J R Soc Med 1983;76:62-70.
4. Navin TR, Juranek DO. Cryptosporidiosis: clinical, epidemi­
ologic, and parasitologic review. Rev Infect Dis 1984;
6:313-27.
5. Nime FA, Burek JD, Page DL, et al. Acute enterocolitis in a
human being infected with the protozoan Cryptosporidium.
Gastroenterology 1976;70:592-8.
6. Meisel JL, Perera DR, Meligro C, et al. Overwhelming watery
diarrhea associated with Cryptosporidium in an immunosup­
pressed patient. Gastroenterology 1976;70:1156-60.
7. Weinstein L, Edelstein SM, Madara JL, et al. Intestinal crypto­
sporidiosis complicated by disseminated cytomegalovirus
infection. Gastroenterology 1981 ;81 :584-91.
8. Soave R, Danner RL, Honig C, et al. Cryptosporidiosis in
homosexual men. Ann Intern Med 1984;100:504-11.
9. Holten-Andersen W, Gerstoft ). Henriksen SA, et al. Preva­
lence of Cryptosporidium among patients with acute enteric
infection. J Infection 1984;9:277-82.
10. Jokipii L, Pohjola S, Jokipii AMM. Cryptosporidium: a fre­
quent finding in patients with gastroiRtestinal symptoms.
Lancet 1983 ;ii:358-61.
11. Wolfson JS, Richter JM, Waldron MA, et al. Cryptosporidiosis
in immunocompetent patients. N Engl J Med 1985;312:
1278-82.
12. Tyzzer EE. A sporozoan found in the peptic glands of the
common mouse. Proc Soc Exp Bioi Med 1907;5:12-3.
13. Tyzzer EE. An extracellular coccidium, Cryptosporidium
muris (gen . et sp. nov.). of the gastric glands of the common
mouse. J Med Res 1910;23:487-509.
14. Hampton JC, Rosario B. The attachment of protozoan para­
sites to intestinal epithelial cells of the mouse. J Parasitol
1966;52:939-49.
15 . Vetterling JM, Takeuchi A, Madden PA. Ultrastructure of
Cryptosporidium wrairi from the guinea pig. J Protozool
1971;18:248-60 .
16. Pohlenz ). Bemrick W). Moon HW, Cheville NF. Bovine
cryptosporidiosis. A transmission and scanning electron mi­
croscopic study of some stages in the life cycle and of the
host-parasite relationship. Vet Pathol 1978;15:417-27.
17. Bird RG, Smith MD. Cryptosporidiosis in man: parasite life
cycle and fine structural pathology. J PathoI1980;132:217-33.
18. Pearson GR, Logan EF. Scanning and transmission electron
microscopic observations on the host-parasite relationship in
intestinal cryptosporidiosis of neonatal calves. Res Vet Sci
1983;34:149-54.
19. Owen RL, Jones AL. Epithelial cell specialization within
human Peyer's patches: an ultrastructural study of intestinal
lymphoid follicles . Gastroenterology 1974;66:189-203 .
20. Wolf JL, Rubin DH, Finberg R, et al. Intestinal M cells: a
pathway for entry of reovirus into the host. Science 1981;
212:471-2 .
21. Owen RL, Piece NF, Apple RT, Cray WD Jr. Phagocytosis and
transport by M cells of intact Vibrio cholera into rabbit
Peyer's patch follicles (abstr). J Cell Bioi 1982;95:446.
GASTROENTEROLOGY Vol. 90, No.3
22. Karnovsky MJ. A formaldehyde-glutaraldehyde fixative of
high osmolality for use in electron microscopy (abstr). J Cell
Bioi 1965;27:137.
23 . Scholtyseck E. Fine structure of parasitic protozoa. New York:
Springer-Verlag, 1979:15-37.
24. Vivier E. Donnees nouvelles sur les sporozoaires. Cytologie­
cycles-system atique. Extrait du Bulletin de la Societe
Zoologique de France 1979;104:345-81.
25. Bye WA, Allan CH, Trier JS. Structure, distribution, and
origin of M cells in Peyer's patches of mouse ileum. Gastro­
enterology 1984;86:789-801.
26 . Madara JL, Bye WA, Trier JS. Structural features of and
cholesterol distribution in M-cell membranes in guinea pig,
rat, and mouse Peyer's patches. Gastroenterology 1984;87:
1091-103 .
27. Bannister LH. The invasion of red cells by Plasmodium. In:
Taylor AER, Muller R, eds. Parasite invasion. Oxford:
Blackwell Scientific, 1977:27-55.
28. Long PL, Speer CA. Invasion of host cells by coccidia. In:
Taylor AER, Muller R, eds . Parasite invasion. Oxford :
Blackwell Scientific, 1977:1-25.
29 . Aikawa M, Komata y, Asai T, Midorikawa O. Transmission
and scanning electron microscopy of host cell entry by
Toxoplasma gondii . Am J Pathol 1977;87:285-96.
30. Aikawa M, Miller LH, Johnson J, Rabbege J. Erythrocyte entry
by malarial parasites . A moving junction between erythrocyte
and parasite. J Cell Bioi 1978;77:72-82 .
31. Aikawa M, Miller LH, Rabbege JR, Epstein N. Freeze fracture
study of the erythrocyte membrane during malarial parasite
invasion. J Cell Bioi 1981;91:55-62.
32. Madara JL. Structural specializations of absorptive cell mem­
branes at filamentous organism-absorptive cell attachment
sites in mammalian small intestine. In: Boedecker EC, ed.
Attachment of organisms to the gut mucosa. Boca Raton, Fla.:
CRC Press, 1984:138-44.
33. Ladda R, Aikawa M, Sprinz H. Penetration of erythrocytes by
merozoites of mammalian and avian malarial parasites. J
Parasitol 1969;55 :63 3-44.
34. Owen RL, Allen CL, Stevens DP. Phagocytosis of Giardia
muris by macrophages in Peyer's patch epithelium in mice.
Infect Immun 1981;33:591-601.
35. Owen RL, Nemanic PC, Stevens DP. Ultrastructural observa­
tions on giardiasis in a murine model. I. Intestinal distribu­
tion , attachment, and relationship to the immune system of
Giardia muris. Gastroenterology 1979;76:757-69 .
36. Miller MF, Martin JR, Johnson p , et al. Erythromycin uptake
and accumulation by human polymorphonuclear leukocytes
and efficacy of erythromycin in killing ingested Legionella
pneumophilia. J Infect Dis 1984 ;149:714-8.
37. Current WL, Haynes TB. Complete development of Crypto­
sporidium in cell culture. Science 1984;224:603-5.
38. Current WL, Long PL. Development of human and calf
Cryptosporidium in chicken embryos. J Infect Dis 1983;148:
1108-13.