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1986 Isolation, in vitro excystation, and in vitro development of Cryptosporidium sp from calves Douglas Byron Woodmansee Iowa State University
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T TA yf.T Dissertation iJ iVll Information Service University Microfilms International A Bell & Howell Information Company 300 N. Zeeb Road, Ann Arbor, Michigan 48106
8627163
Woodmansee, Douglas Byron
ISOLATION, IN VITRO EXCYSTATION, AND IN VITRO DEVELOPMENT OF CRYPTOSPORIDIUM SP. FROM CALVES
Iowa State University PH.D. 1986
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University Microfilms International
Isolation, in vitro excystation, and in vitro development of
Cryptosporidium sp. from calves
by
Douglas Byron Woodmansee
A Dissertation Submitted to the
Graduate Faculty in Partial Fulfillment of the
Requirements for the Degree of
DOCTOR OF PHILOSOPHY
Department: Zoology Major; Zoology (Parasitology)
Approved:
Signature was redacted for privacy. In Charge of Maj^r Work
Signature was redacted for privacy. For the Major Department
Signature was redacted for privacy. For the Graduate College
Iowa State University Ames, Iowa
1986 ii
TABLE OF CONTENTS
Page
GENERAL INTRODUCTION 1
Explanation of Dissertation Format 3
LITERATURE REVIEW 4
Historical Aspects 4
Taxonomy 10
Life Cycle and Morphology 15
Diseases Associated with Cryptosporidium 29
SECTION I. DEVELOPMENT OF CRYPTOSPORIDIUM SP. IN CULTURED CELLS 46
Abstract 46
Introduction 46
Materials and Methods 47
Results 49
Discussion 51
Literature Cited 52 iii
SECTION II. AN IN VITRO STUDY OF SPORULATION IN CRYPTOSPORIDIUM SP. 71
Abstract 71
Introduction 71
Materials and Methods 73
Results 74
Discussion 75
Literature Cited 75
SECTION III. TAUROCHOLIC ACID AFFECTS MOTILITY AND MORPHOLOGY OF CRYPTOSPORIDIUM SPOROZOITES IN VITRO 84
Abstract 84
Introduction 84
Materials and Methods 85
Results 88
Discussion 89
Literature Cited 91 iv
SECTION IV. STUDIES OF IN VITRO EXCYSTATION OF CRYPTOSPORIDlUM SP. FROM CALVES 106
Abstract 106
Introduction 106
Materials and Methods 107
Results 112
Discussion 115
Literature Cited 118
SECTION V. TECHNIQUES FOR THE RECOVERY OF CRYPTOSPORIDIUM OOCYSTS AND SPOROZOITES FROM CALF FECES 142
Abstract 142
Introduction 142
Materials and Methods 143
Results and Discussion 146
Literature Cited 149
GENERAL SUMMARY 151
LITERATURE CITED 154
ACKNOWLEDGMENTS 184 1
GENERAL INTRODUCTION
The genus Cryptosporidium consists of intracellular parasitic protozoa which infect the apical portions of epi thelial cells of several organs. The implication of infec tion by Cryptosporidium as a cause of disease (particularly diarrhea) has attracted substantial interest from research ers in human and veterinary medicine. In cattle and sheep, the infection appears to be a cause of the neonatal diarrhea syndrome known as scours. In humans, infections are associ ated with severe, protracted diarrhea in immunosuppressed patients and with less severe, self resolving diarrhea in immunologically normal people (43a, 199).
Most of the recent research on Cryptosporidium has been
done by pathologists "and clinicians rather than parasitolo
gists. Therefore, the research emphasis has been heavily
weighted toward studies of the pathological, clinical and
epidemiological features of the disease rather than toward
the taxonomy, physiology and comparative biology of the
parasite. As a result of this emphasis, there is strong
evidence that Cryptosporidium is a primary pathogen and the
clinical aspects of the disease are well documented. We
have a poorer understanding of the taxonomy, life history
and basic biology of the parasite. 2
Studies of the biology of Cryptosporidium in comparison to other coccidian parasites are few, despite a number of unusual features of Cryptosporidium which are quite inter esting. The most common reason given for this neglect is that Cryptosporidium is difficult to work with. It is the smallest coccidian, with oocysts only about half the size of most other species. Furthermore, growth of the parasite in cultured cells (difficult for any coccidian) has not yet been achieved in a manner conducive to physiological studies. Finally, isolation of Cryptosporidium life cycle
stages from in vivo sources has been far less efficient than
the isolation of similar stages of other species.
This dissertation reports efforts to address some of
these problems. Specifically, efforts to grow Cryp
tosporidium in cell culture, an examination of the sporula
tion phase of the life cycle, a study of coccidian excysta-
tion using Cryptosporidium as a model organism, and new
techniques for the isolation of oocysts and sporozoites are
reported. The variety of topics approached in this disser
tation is a reflection of the breadth of the work that still
needs to be done with this sometimes fascinating, and some
times frustrating organism. 3
Explanation of Dissertation Format
This dissertation is written in the alternate format.
Five papers are presented in addition to a general introduc tion, a general literature review, and a general summary.
References for the general introduction and literature re view follow the general summary. Portions of the paper en titled "Development of Cryptosporidium sp. in cultured cells" were published in the Proceedings of the Fourth
International Symposium on Neonatal Diarrhea, 1983, Veteri nary Infectious Disease Organization, Saskatoon, Sask.,
Canada, pp. 306-319. The paper entitled "An in vitro study of sporulation in Cryptosporidium sp." has been accepted for publication in the Journal of Parasitology. All parts of this dissertation have been written to conform to the style required by the Journal of Protozoology. 4
LITERATURE REVIEW
Historical Aspects
The genus Cryptosporidium was first erected by Tyzzer in 1907 in a preliminary description of Cryptosporidium muris (172); a complete report followed in 1910 (173). Tyz zer found large numbers of protozoans infecting the gastric glands of laboratory mice- He thoroughly described these parasites with light microscopy using both stained and un stained preparations, and experimentally infected weanling mice with mucosal scrapings from naturally infected mice.
His work demonstrated that these parasites were not develop mental stages of other murine protozoan parasites and that reports by other investigators (34, 194) describing small developmental stages of Eimeria (Coccidium) falciformis may have been misinterpreted observations of Cryptosporidium.
Tyzzer recognized Cryptosporidium muris to be a sporozoan parasite. He used the nomenclature established for sporozoans to describe the life cycle stages: schizonts for the asexual developmental stage, micro- and macrogametes for the sexual stages, oocysts for the resistant stage which
develops after fusion of gametes, and sporozoites for the
individual parasites which develop by asexual reproduction
within the oocyst. The name Cryptosporidium is derived from
Greek roots and was intended to "signify that it is a 5
sporozoan in which spores are indistinguishable or absent in the oocyst..." (173).
In 1912 Tyzzer described a second species, Cryp tosporidium parvum (174). The two species were differen tiated on the basis of site specificity, C. muris infecting the gastric glands and C. parvum infecting the small intes tine. Cross transmission experiments were performed to sup port the separation. The developmental stages of C. mur is tended to be larger than similar stages of C. parvum; for example, the oocysts of C. muris measured 7x5 jam while the oocysts of C. parvum were more spherical and measured 4.5 ;um at their greatest diameter. Both species were attached to the apical portion of epithelial cells by a specialized zone of attachment. In C. muris, this structure was frequently visible on parasites which had detached from epithelial cells in stained smear preparations (173), but C. parvum rarely detached, and the attachment zone was much less dis tinct (174). Neither C. muris nor C. parvum infections were associated with disease in the mice.
Tyzzer's work is important because it was the first set of published observations of Cryptosporidium and also be cause the quality of the work was so high. His light
microscopy observations are still the most complete in the
literature, and most of them have been confirmed by modern
methods. 6
Since Cryptosporidium was not initially recognized as a cause of disease, interest in the genus was limited to, as
Levine (96) put it, "serindipitists and taxonomists." Most of the papers published from 1907 to 1975 were descriptions of new species. During these years, the concept of strict host specificity within the Coccidia was dogma. It was, and to a certain extent still is, common practice to create a new species designation each time a coccidian parasite was discovered in a new host. Tables were published in which the genera of the Coccidia were grouped according to their oocyst morphology (see pp. 92-93 of reference 86 for exam ple). If a coccidian oocyst was found in a fecal sample, the table could be used to assign it to a genus. If a re view of the literature revealed that no members of that ge nus had previously been described in that host species, then a new species could be erected. Strict host specificity was usually assumed but cross-transmission experiments were often not performed. In retrospect, it is clear that such
practices led to an inflated list of species within the
genus Cryptosporidium. Levine (9 6) has recently reviewed
the species erected within the genus.
The first serious adjustment to the list of species was
made by Vetterling et al. in 1971 (189). He pointed out
that in some cases, species described on the basis of oocyst
morphology were probably misinterpreted observations of free 7
Isospora-like sporocysts. Isosporan sporocysts and Cryp tosporidium oocysts both contain four free sporozoites, so it would be natural to assign such sporocysts to the genus
Cryptosporidium if cyst morphology were the only criterion used. Vetterling stated that species named on the basis of cyst morphology alone should be considered invalid.
A second major development was the discovery by Tzipori and his colleagues in 1980 (177) that at least some isolates of Cryptosporidium could be readily transmitted to various host species; demonstrating that strict host specificity can not be assumed in the case of Cryptosporidium. These find ings raised doubts about the validity of most of the remain ing species. As a result of these problems, most authors
have been reluctant to use a species name to refer to the
isolates they work with. Thus, one often sees the parasite
referred to as Cryptosporidium sp., Cryptosporidia or just
Cryptosporidium.
The first association of Cryptosporidium infections
with disease came in 1955 when Slavin (159) reported mild
diarrhea causing unthriftiness and occasional death in in
fected turkey polts. In 1966, another report described in
fection associated with microscopic lesions in the small
intestine of guinea pigs (81). In the early and mid 1970s,
reports of disease associated with Cryptosporidium infec
tions began to appear with more frequency. Among them were 8
reports describing an association between Cryptosporidiuin infection and diarrhea in calves (121, 138, 153). A group
of papers published in the late 1970s (114, 117, 136) showed that Cryptosporidium is probably common in calves with scours. These papers changed the emphasis of research on
cryptosporidiosis in calves away from case reports and to
ward critical attempts to demonstrate that Cryptosporidium
was a primary pathogen. This objective appears to have been
achieved by recent work with germ-free animals and purified
inocula (64, 187). In these studies, Cryptosporidium infec
tions resulted in clinical disease in the absence of ex
traneous agents.
The first report of infection of a human came in 197 6
when Nime et al. (120) observed Cryptosporidium in a three
year old girl with diarrhea. Other reports followed quickly
and it became clear that Cryptosporidium was associated with
diarrheal disease in humans, particularly in immunocompro-
mized individuals. Late in 1982, the Centers for Disease
Control published a report of 21 cases of protracted, in
curable diarrhea caused by Cryptospordium in patients suf
fering from the acquired immunodeficiency syndrome (AIDS)
(31). Reports of Cryptosporidium in AIDS patients began to
appear in the standard medical literature in 1983 and have
continued with some frequency until the present time. 9
In 1983 Current and co-workers (43a), in addition to describing one of the first cases of cryptosporidiosis in an
AIDS patient, proposed that cryptosporidiosis was a cause of diarrhea in immunologically normal humans. Prior to this paper, reports of the disease in normal individuals were relatively infrequent. Animal reservoirs were implicated as
being important in the epidemiology of the disease (43a) and
the suggestion was made that it should be considered a
zoonosis (155). This concept was disputed by Casemore and
Jackson (30) who, based on observations of cryptosporidosis
in a "semi-rural population" in Wales, hypothesized that
person to person transmission was probably more common than
zoonotic transmission. This hypothesis has received strong
support in recent papers (169, 199).
Research is continuing into many aspects of Cryp
tosporidium and cryptosporidiosis. These areas include
efforts to clarify the status of the species within the ge
nus, the establishment of suitable laboratory models for
cryptosporidiosis (including both in vitro and in vivo mod
els), studies of the pathogenesis of the.disease, and
studies of immune responses to the parasite. Finally, an
important area of current research is the search for an ef
fective treatment. To date, cryptosporidiosis has defied
chemotherapy despite repeated attempts with a wide variety 10
of drugs (see 175 for review). The lack of effective treat ment results in a grave prognosis for AIDS patients with cryptosporidiosis (43a).
Taxonomy
The genus Cryptosporidium Tyzzer, 1907 is classified as
follows: phylum Apicomplexa Levine, 1970; class Sporozoasida
Leuckart, 1879; subclass Coccidiasina Leuckart, 1879; order
Eucoccidiorida Leger & Duboscq, 1910; suborder Eimeriorina
Leger, 1911; family Cryptosporidiidae Leger, 1911 (95). The
characteristics of each of these taxa have recently been
described in detail (96, 98) and will not be reviewed here.
Among the Eimeriorina the family Cryptosporidiidae ap
pears to be most closely related to the Eimeriidae, a family
which includes the genera Eimeria, Isospora, and several
others. The two families are similar to each other in terms
of their basic life cycles and morphology. They differ in
that the Cryptosporidiidae develop just beneath the host
cell membrane while the Eimeriidae develop in the host cell
cytoplasm proper. Levine (97) moved the genus Epieimeria
from the Eimeriidae to the Cryptosporidiidae on the basis of
its site of development within host cells, illustrating the
closeness of the two families.
The confusion surrounding the validity of named species
within the genus Cryptosporidium has already been alluded 11
to. The work of Vetterling et al. (189) and Tzipori et al.
(177) cast serious doubt on almost all of the species named within the genus through about 1980, and Tzipori et al. went so far as to suggest that Cryptosporidium was a single-
species genus. Levine (96) reviewed the literature through
1982 and concluded that four species should be recognized as
valid: Cryptosporidium muris Tyzzer, 1907; C. crotali, Trif-
fit, 1925; C. meleagridis Slavin, 1955; and C. nasorum
Hoover, Hoerr, Carlton, Hinsman and Freguson, 1981. Essen
tially, Levine lumped all those species described from mam
mals into C. muris, all those described from snakes into C.
crotali, all those from birds into C. meleagridis, and the
one species from fish was left as C. nasorum.
Levine justified this scheme on the basis that cross-
transmission experiments, when they had been performed, had
tended to show transmission within host classes rather than
across them. Levine's scheme, however, does not account for
all of the experimental evidence. Tzipori et al. (177) re
ported tranmission of Cryptosporidium from calves to one day
old chickens, an exception to the rule that these parasites
do not infect across host class boundaries. Current et al.
(43b) were unable to transmit an isolate from chickens to
quail or turkeys. Unpublished studies (D. B. Woodmansee, I.
Pavalasek and J. F. L. Pohlenz, National Animal Disease Cen
ter, Ames, lA) have demonstrated transmission of an isolate 12
of Cryptosporidium from turkeys to calves and mice. As more experiments are performed more exceptions are likely to be found, and Levine's scheme (or at least the arbitrary dis tinction between C. muris and C. meleagridis) may become untenable.
Tyzzer (174) separated C. parvum from C. muris on the basis of site specificity, not host specificity. In light of Tyzzer's careful description of the morphologic differen ces between the two species and the cross-transmission ex periments which were performed, proposals to synonymize them
(96, 177) would seem unreasonable. It would appear that only rediscovery of C. muris as described by Tyzzer will clarify this point completely. Upton and Current (188) claimed to have rediscovered C. muris upon observing large
Cryptosporidium oocysts in the feces of cattle. They felt that these large oocysts were C. muris oocysts, and that the
smaller forms seen in most other studies of Cryptosporidium
in cattle were oocysts of C. parvum. While it is clear that the large oocysts they observed appeared different from the oocysts most commonly seen in calf feces, it is not certain
that they were actually C. muris. To be sure of that, it
would be necessary to transmit the parasites to mice, demon
strate infection of the gastric glands, and show that other
stages of the life cycle, not just the oocyst, are similar
to Tyzzer's original description. Reports of the discovery 13
of Cryptosporidium-like parasites infecting the abomassum of cattle which were shedding the large form of the oocyst in their feces (B. C. Anderson, Caldwell Veterinary Teaching
Center, Caldwell, ID, unpublished) encourages one to believe that Upton and Current may be essentially correct. One can only hope that the next "rediscovery" of C. muris will be more convincing than the first.
The case of Cryptosporidium crotali is another case in which Levine appears to be mistaken. The species was
described by Triffit (171) on the basis of oocyst morphol ogy. The oocysts measured at least 10 and up to 12.5 jam in
diameter, far larger than any known Cryptosporidium oocyst.
As pointed out by Vetterling et al. (189), oocyst morphology
is not adequate for the description of a species of Cryp
tosporidium. Judging by their size, the cysts Triffit saw
were far more likely to be sporocysts of Sarcocystis or per
haps, Isospora than oocysts of Cryptosporidium. At this
time the data are insufficient to determine the true
relationship of Cryptosporidia from snakes to Cryptosporidia
from other animals. The same statement is also true for the
cryptosporidian observed in a fish (71).
While the species within the genus Cryptosporidium are
far from being resolved, it is possible to make some hypoth
eses to frame future work in this area. First, it appears
that one species of Cryptosporidium is resposible for most 14
of the infections which result in clinical disease in laam- mals. Abundant examples of cross transmission experiments and case studies implicating infection across species lines now exist (39, 97, 188). This parasite matches, in most
respects, the description given by Tyzzer (174) for Ç. par-
vum and many investigators in the field believe that it
should be referred to as such (188). It is reasonable to
hypothesize that the species described as C. muris is valid
and efforts should be made to rediscover this parasite and
confirm the hypothesis.
Recent, unsuccessful attempts to infect mammals with
certain isolates of Cryptosporidium from chickens (43b) and
vice versa (125) have indicated to some that differences
between Cryptosporidia of birds and mammals great enough to
deserve species ranking exist. Exceptions also exist
however (177), and until more studies are performed, it may
be best to take the position that all new isolates of Cryp
tosporidia are C. parvum until proven otherwise. Proof may
take the form of mutually exclusive cross-transmision ex
periments, distinctive and consistent morphological or
developmental differences, or perhaps distinctive and con
sistent differences in isozyme or nucleotide restriction
patterns. 15
Life Cycle and Morphology
Sporozoites
The life cycle of coccidian parasites is initiated when sporozoites are released from oocysts and invade hosr cells.
The sporozoites of Cryptosporidium have been variously described as boomerang (173), banana (76, 78), or comma (42) shaped. The cytoplasm stains blue and the nucleus red with
Giemsa's stain (76). The nucleus appears rod-shaped in stained smears (173, 174) but round in fresh preparations observed with interference contrast microscopy (42). Re ported sizes of these organisms vary from 4-6 x 0.5-1.2 jum
(42, 78, 145, 174, 188). Sporozoites believed to be those
of Cryptosporidium muris are larger and have been reported
to measure 11.1 x 1.0 aim (173, 188).
The ultrastructure of Cryptosporidium sporozoites is
similar to that of other coccidians (for a review of coc
cidian ultrastructure see 33). Features include the pelli
cle, rhoptries, micronemes, a nucleus, and ribosomes (76,
78). Examination of the surface of sporozoites by scanning
electron microscopy shows numerous, randomly scattered pits,
the function of which is unknown (145).
Host cell penetration
Discussion of host cell penetration is complicated by
claims that Cryptosporidium is actually extracellular and 16
thus does not invade host cells at all (135, 173, 174). The bulk of the data support the idea of intracellular develop ment, and examination of papers which claim the contrary show that the data are not inconsistent with the intracellu lar hypothesis. Since mechanisms of host cell invasion ap pear to be similar for both sporozoites and merozoites, the following description is based on data from both forms.
Vetterling et al. (190) likened invasion to inverting the finger of a rubber glove; membrane from the host cell
moves up the invading sporozoite and seals at the top, leav
ing the parasite covered with a double membrane of host ori
gin called the parasitophorous envelope (135). Unlike other
apicomplexan parasites, Cryptosporidia do not penetrate into
the host cell cytoplasm proper, but stop such that only the
anterior end of the organism interfaces with the host cell
while the posterior end remains in the lumen of the infected
organ. Several transmission electron micrographs of zoite
invasion have been published (19, 42, 74, 76, 78, 107, 130,
190). The source of available host membrane required by
this model is unknown, but one possibility is that microvil
li adjacent to the site of attack fuse and donate the
membranes covering them to the envelope (57, 74, 130).
Tyzzer (174) observed movement of a granule from the
cytoplasm of the sporozoite to the zone of attachment during
invasion. Other investigators have described vacuolation of 17
the anterior end of sporozoites during penetration (42, 76,
190). It is likely that these observations represent empty ing of rhoptries and micronemes as seen in penetrating
Eimeria sporozoites (79).
Trophozoites
After penetration, sporozoites round up and are called trophozoites or uninucleate raeronts. The host-parasite relationship and much of the ultrastructure is identical in all the attached stages of the life cycle, so much of the description of the trophozoite will carry over to the later stages. Trophozoites are round to oval with blue cytoplasm and a reddish nucleus after Giemsa staining (159, 173, 174).
They grow as they mature (58), and reported sizes range from
1.5-5.0 |im in diameter (11, 42, 78, 159, 174).
The parasite is covered with the parasitophorous enve lope. The outer membrane of the envelope is continuous with the apical membrane of the epithelial cell (58, 76, 78, 105,
190) and is covered with a glycocalyx (57, 58, 105, 135).
Beneath the envelope is a space, known as the
parasitophorous vacuole. The trophozoite itself is covered
by a three membrane pellicle, the inner two membranes of
which regress as the parasite grows until they only appear
at the top of the cell (42, 78, 105, 190). Freeze fracture
studies of the trophozoite pellicle show that the outer two 18
membranes have low numbers of intramembranous particles while the inner membrane is rich in them (105). This find ing is typical for the pellicles of other coccidians (33).
At the base of the parasite is an unusual structure called the attachment organ (173) or feeder organelle (135).
It is a network of membranous folds which forms the inter face between the parasite and the host cell. It is believed that during early development of the trophozoite, the host cell membrane separating the parasite pellicle from the host cell cytoplasm disintegrates (42, 58, 105) and the outer
pellicle membrane hypertrophies to form the organelle (105).
While its function has never been proven, authors have fre
quently speculated that it is involved in nutrition of the
parasite (135, 173, 174, 190).
The edges of the feeder organelle are defined by a
structure called the annular ring (135). One author
described this structure as a junctional complex (42), while
another described it as a pentilaminar membrane fusion site
(105). Beneath the feeder organelle is an electron dense
band (58, 76, 130) which was once thought to be a remnant of
the host cell plasma membrane (58) but this interpretation
is no longer in favor (105). Together, the annular ring and
dense band form a stable and strong attachment between the
the parasite and the host cell. 19
The cytoplasm of young trophozoites contains only a nucleus with a large nucleolus, Golgi anlagen and ribosomes
(76, 78, 190). Mitochondria are believed to be absent from all stages of the life cycle (42, 190). As the trophozoite grows, rough endoplasmic reticulum develops near the nucleus and expands until it fills the entire cytoplasm. The feeder organelle also increases in size as the trophozoite matures
(42, 78, 107, 190).
Trophozoites are indistinguishable from gametes by scanning electron microscopy. These forms show fluting of the parasitophorous envelope around the base (74, 129, 130).
It has been suggested that these folds represent fusion of microvilli to the envelope (57, 74) but it is also possible that they are processing artifacts (129, 200).
Meroqony
After a mature trophozoite begins nuclear division it is called a meront or schizont. During merogony the parasites reproduce by multiple fission and the population of parasites in the tissue grows rapidly. The number of offspring (known as merozoites) per meront and the number of types (also called generations) of meronts which occur
varies among the species of the Coccidia (86).
In the case of Cryptosporidium, multiple meront types
have been described. Type I meronts give rise to eight 20
merozoites while Type II meronts give rise to four merozoites (42, 110, 135, 189). The two types are also dif ferentiated by the arrangement of the merozoites within the meront. Type I merozoites lie randomly in the parasitophorous envelope while Type II merozoites are ar ranged like the sections of an orange, with their anterior ends oriented away from the feeder organelle (42). Some authors have described only Type I meronts (19, 76, 78, 159,
173, 174) and there is some debate whether or not the Type
II meront is truly a life cycle stage. A third meront type
(Type III) has also been described on one occasion (43b).
Nuclear division occurs to yield a number of nuclei equal to the number of merozoites which will ultimately be formed (19, 42, 76, 78, 173, 174, 189). Vetterling et al.
(190) failed to observe centrioles, spindles, plaques or intranuclear filaments during nuclear division. After
nuclear division, the nuclei move to the periphery of the cell in preparation for cytokinesis (42, 173, 174, 189).
Merozoites bud off from anterior to posterior (190).
Each merozoite has a three membrane pellicle despite the
fact that the inner two membranes are limited in the
trophozoite (42, 76, 105, 190). A nucleus, Golgi apparatus,
and rough endoplasmic reticulum are included in each
merozoite (42, 76, 190). The apical complex forms rapidly
and it is not unusual to find ultrastructurally complete 21
merozoites still attached to the feeder organelle by a small peduncle (42, 135, 190). Most of the meront's cytoplasm
becomes incorporated into the merozoites. When cytokinesis
is complete, the merozoites lie free in the parasitophorous
vacuole and the remainder of the meront, together with the
feeder organelle, is left as a residual body covered with a
single unit membrane (190).
Scanning electron microscopy of meronts demonstrates
the delicate nature of the parasitophorous envelope. The
envelope may collapse around the merozoites leaving their
outlines clearly visible (200) or it may be torn away, leav
ing the merozoites exposed (135).
Like trophozoites, meronts vary in size. Reports of
meront diameter range from 3-8 ;im but reports of 4-5 jom are
most common (42, 52, 76, 110, 159, 174, 190). Since, mea
surements are affected by fixation and staining techniques
(110), measurement of live specimens using interference con
trast microscopy (42) may be the best method.
Vetterling et al. (189) observed Type I meronts 7-9
days post inoculation (p.i.) and Type II meronts from 11-14
days p.i. in the guinea pig intestine. Current and Reese
(42) observed early Type I meronts at 12 h p.i. and mature
Type I meronts at 16 h p.i. in the intestine of mice while
Type II meronts were first observed at 16 h p.i. Both types 22
were seen up to 9 days p.i. Current and Haynes (40), work ing with infected cell cultures, observed Type I meronts from 12-96 h p.i. and Type II meronts from 24-144 h p.i.
Merozoites
The morphology of merozoites is similar to the morphol ogy of sporozoites. They are banana shaped (42, 78) and have been reported to measure 0.7-1.2 x 2-8 jom (42, 52, 76,
78, 159, 173, 189). The nucleus is spherical to subspheri-
cal and subterminal (42, 76, 159, 173, 174). Tyzzer
described a granule in the anterior portion of the merozoite
identical to the granule found in the sporozoite (173, 174).
Merozoites move by gliding and flexing (42).
Rhoptries, micronemes and a conoid have been observed
by electron microscopy (19, 42, 76, 78, 190). Polar rings
have been reported (19, 190) but a recent paper has inter
preted these structuras as preconoidal rings (42). The pel
licle consists of two membranes (42, 78) and may not cover
the anterior end of the merozoite (42). Micropores and sub-
pellicular microtubules, conspicuous features of other coc-
cidian zoites (33), are absent from Cryptosporidium
merozoites (19, 42, 190), but microtubules supporting ducts
leading from rhoptries have been described (42). A Golgi
apparatus and rough endoplasmic reticulum also occur (19,
76, 78, 190). 23
Current and Reese (42) have described differences between merozoites arising from Type I and Type II meronts.
They describe Type I merozoites as slightly shorter and slightly wider than Type II and claim that Type I merozoites may give rise to either Type I meronts or Type II meronts while Type II merozoites only give rise to gametes.
Microqamonts and microqametes
Microgamonts give rise to microgametes, the male ga mete. They are shaped similar to immature meronts and mea sure 2-9-6 Jim (42, 52, 57, 110, 159, 173). They have up to
16 dense, peripherally arranged nuclei, ribosomes, endoplas mic reticulum and vacuoles (19, 42, 57, 78, 159, 173, 174,
189).
The nuclei and a small amount of cytoplasm bud off to form the microgametes, leaving a large residuum (19, 57, 78,
173). Microgametes have been reported to measure 0.2-0.4 x
9.5-1.1 jam (42, 57, 78, 189). They have been described as bullet shaped (42), wedge shaped (57), or tubular (78), but all descriptions mention an expanded and flattened anterior end and a pointed posterior end. The pellicle is a double membrane (57, 78, 190) and there are no flagella (19, 42,
76, 173). On two occasions authors described tubular
mitochondria within microgametes, (19, 57) but the photo
graphs are unconvincing and have been challenged (42). 24
The microgamete interacts with the female or macroga- mete by its anterior end (57). This portion, called the adhesion zone, is composed of granular osmiophilic material, polar rings and several associated microtubules (57, 78).
The mechanisms by which microgametes recognize and interact with macrogametes are unknown. Microgametes are motile and have been observed to exibit "jerky foward gliding move ments" (42).
Macrogametes
Macrogametes are the female sexual stage. Reported sizes range from 3-7 ;iim in diameter (11, 42, 52, 57, 76, 78,
110, 159, 174, 189). The cytoplasm of these cells stains blue with Giemsa and takes on a characteristic spongy ap pearance due the extraction of material from numerous granules (57, 159, 173, 174). These granules are stainable with Lugol's solution (76, 173), indicating the presence of carbohydrate.
Ultrastructural studies of macrogametes reveal a cyto
plasm containing polysaccharide granules. Similar granules
in Eimeria oocysts have been shown to contain amylopectin
(150). In addition to these granules, the cytoplasm con
tains wall forming bodies. These membrane-bound bodies,
typical for coccidian macrogametes, contain material which
will later be laid down to form the oocyst wall (33). 25
Scholtyseck et al. have described two types of wall forming bodies in Eimeria, WFl and WF2 (154). Some authors claim that both types are present in Cryptosporidium (42, 78) while others claim that only WFl occur (57, 190). Rough endoplasmic reticulum and large membrane bound vacuoles, thought to contain lipid, also occur (57, 75, 78, 135, 189).
Fertilization and Sporulation
Fertilization of macrogametes by microgametes to form zygotes has been observed with both light and electron microscopy (57, 159, 173, 174). After fertilization, the wall forming bodies move to the periphery of the macrogamete and release their contents into the parasitophorous envelope
(42). As a result, the cytoplasm of the zygote becomes sep
arated from the feeder organelle by the newly formed oocyst
wall and the zygote lies free in the parasitophorous vacuole
(11, 42). This stage is called the unsporulated oocyst.
Oocysts complete their development and form sporozoites
by a form of multiple fission known as sporulation. In
other coccidians, sporulation is believed to be a meiotic
event (27). Sporulation of Cryptosporidium may occur in the
parasitophorous envelope (11, 42, 76) but some authors have
suggested that substantial sporulation occurs in the intes
tinal lumen (173, 174) or outside of the host (3). 26
Spomlated Oocysts
Sporulated oocysts are passed in feces and may infect a new host if they are ingested. They are resistant to en vironmental stress, particularly chemical disinfectants (9,
26, 126) and may maintain viability for several months if
kept cool and wet (42). Oocysts of C. muris measure 5-5.6 x
7-7.4 jam (173, 188) while other cryptosporidian oocysts
(probably C, parvum) measure 3.3-5.0 x 4.0-5.2 jam (42, 76,
145, 159, 174, 188).
The time from infection to the onset of oocyst shedding
(the prepatent period) and the duration of oocyst shedding
(the patent period) has been reported for a number of ex
perimental hosts (8, 40, 42, 61, 76, 113, 115, 116, 177,
178, 183, 186, 187). Prepatent periods are commonly about
three days and patent periods usually 10-14 days.
The ultrastructure of mature oocysts is difficult to
study because they become severely distorted by conventional
electron microscopic techniques (11, 42). It is generally
agreed that mature cysts contain four sporozoites, and a
membrane bound residuum filled with polysaccharide granules
(42, 76, 78, 135, 145, 173, 174, 188).
The oocyst wall is made up of an inner, electron dense
layer and an outer, electron lucent layer (42, 78). Its
structure has recently been described in detail (144). The 27
wall is interrupted by a well defined suture line which ex tends roughly 1/3 to 1/2 the way around the oocyst (42, 145,
188). Dltrastructurally, the suture consists of a series of alternating electron dense and electron lucent bands (144).
Some investigators have claimed the existence of a second type of oocyst, the thin-walled oocyst. This form was first described by Current and colleagues from ex perimentally infected chicken embryos (41) and mice (42).
The four sporozoites are surrounded by a membranous cyst wall which is easily ruptured. It was proposed that autoin fection as a result of release of sporozoites into the in testinal lumen from thin-walled oocysts is an important mechanism by which Cryptosporidium infections become persis
tent in immunocompromized hosts (42).
Some authors have argued that the cyst stage of Cryp
tosporidium should be referred to as a sporocyst rather than
an oocyst (21). This position has not gained general accep
tance (42). Levine's uniform terminology for apicomplexans
(94) defines the oocyst as "A cyst formed around a zygote."
and the sporocyst as "A cyst formed within the oocyst...."
Since only one cyst wall develops around the Cryptosporidium
zygote (42, 76, 173, 174) that cyst is, by definition, an
oocyst. This interpretation is consistent with the original
definition of the genus (173). 28
Excystation
Excystation is the process by which sporozoites escape the oocyst so that they may infect new host cells. Excysta tion of eimerian oocysts is well studied and some of the biochemical mechanisms have been worked out (149, 191).
The suture in the oocyst wall of Cryptosporidium is the structure through which sporozoites escape. Reduker et al.
(144) have described the dissolution of the suture in response to excystation stimuli. The data suggest that excystation proceeds from the inside out. The first changes are a loss of an internal ridge directly beneath the suture.
This is followed by separation of the internal aspects of the bands making up the suture. Finally, the outermost part of the inner oocyst wall and the outer wall rupture. Scan ning electron microscopic observations show a depression in the suture prior to the formation of the cleft-like opening through which the sporozoites escape (145).
Studies of excystation in response to various stimuli
have established that the excystation process in Cryp
tosporidium may not be identical to excystation of
eimerians. It appears that Cryptosporidium oocysts do not
require activation by reducing conditions and CO2 (49,
143b). Furthermore, it is possible for Cryptosporidium
oocysts to excyst in the absence of host digestive enzymes 29
and bile salts (49) but this may require a chemical pre- treatment (143b). These observations led Payer and Leek
(49) to hypothesize that excystation in Cryptosporidium is
mediated by an internal enzyme system. Such systems have
been proposed for Eimeria but never proven to exist (191).
Diseases Associated with Cryptosporidium
Cryptosporidiosis in humans
Clinical Aspects The clinical presentation of in
testinal cryptosporidiosis in humans is variable. In the
case of non-immunosupressed persons, the disease is a self
limiting, non-life threatening diarrhea. Case reports have
described diarrhea with the following characteristics in
various combinations: usually mild (22, 166) but sometimes
severe (16, 120, 133, 146), watery (16, 22, 51, 87, 102,
120, 180), foul smelling (148), green (51), and sometimes
containing mucus (133, 201) and/or blood (120, 133).
Reports of the duration of diarrhea in non-
immunosuppressed humans vary from 1 day (43a) to 5 months
(75), but the majority of cases last 1 to 2 weeks (14, 22,
59). Cases of people shedding oocysts without diarrhea have
occurred (43a, 69, 164) and shedding may continue for a time
after the diarrhea resolves (14, 69). In some cases the
diarrhea is discontinuous (59). 30
Abdominal pain, anorexia, vomiting, and fever in addi tion to diarrhea are commonly reported (14, 69, 201). De hydration (16, 166), headache (102), malaise (43a, 87, 10 2,
146, 148, 180), fatigue (87, 180), flatulence (69), hot and cold flushes (5, 180) and constipation (146) are less com mon. Diarrhea tends to be the last of the clinical signs to resolve (43a, 59, 180).
Cryptosporidiosis of immunosuppressed people is usually severe, persistent and often life threatening. The disease is most frequent in patients with AIDS (31) but also occurs in patients with congenital hypogammaglobulinemia (43a, 91,
160), immunodeficiencies of unclear origin (89, 165) and persons undergoing immunosuppressive chemotherapy (109, 111,
193).
Clinical signs are qualitatively similar to those found in immunologically normal people but are usually more severe and often complicated by the presence of other diseases.
Mean fecal output of 21 patients was reported to be 3.6
liters/day with one patient excreting 17 liters/day (31).
Diarrhea and oocyst shedding fail to resolve and continue
until the death of the patient in most cases. Exceptions to
this scenario including spontaneous resolution of diarrhea
have been known to occur (18, 160, 2 02) and it is common for
patients taking immunosuppressive drugs to clear the infec
tion if taken off of the drugs (109, 111). Supportive 31
therapy may keep persistently infected patients alive indef initely but malnutrition as a result of severe diarrhea has been listed as contributing to death in some instances (89,
160, 165).
Pathology Knowledge of the pathology of human cryp- tosporidiosis has come primarily from the study of intes tinal biopsy specimens. Changes observed in these specimens include shortened villi (20, 89, 91,109, 165, 193), in creased crypt depth (20, 91, 109, 165) and increased numbers of inflammatory cells in the lamina propria (7, 19, 91, 109,
165, 193). Changes at the cellular level are cuboidal epi
thelial cells (19, 91, 109), disrupted microvilli (19, 91,
120, 193, 165), and peaks in the cytoplasm of the cells at
the point of parasite attachment (91, 93).
The parasites can colonize a number of sites within the
digestive tract including the stomach (7, 20), duodenum (7,
19, 93, 165), jejunem (20, 89, 91, 93, 109, 165, 193), ileum
(7, 19, 109), colon (19), and rectum (19, 120) as well as
extra-intestinal sites including the gall bladder (20, 91,
134), respiratory tract (23, 89) and pancreatic duct (89).
It is difficult to determine the relative importance of the
various sites because biopsy specimens are often taken from
only one site in a given patient and in no case do the au
thors give an objective evaluation of the number of
parasites in the samples. 32
Little is known of the pathogenesis of cryp- tosporidiosis but some authors have speculated that the di arrhea is of the secretory type based on its sometimes se vere, watery nature and on the outcome of preliminary pathophysiological tests (7, 89). The histopathology of the intestine, with its characteristic villus atrophy, crypt hyperplasia, and colonic lesions suggests that malabsorption may also contribute to the diarrhea.
Diagnosis The earliest cases of cryptosporidiosis in humans were diagnosed by microscopic examination of in testinal biopsy specimens (120, 109). Soon thereafter, techniques developed in veterinary medicine for finding oocysts in feces were applied to human cases. Tzipori et al. (180) used Giemsa stained fecal smears as developed by
Pohlenz et al. (136). Reese et al. (146) and later Current et al. (43a) used Sheather's sucrose flotations (a standard technique for diagnosis of parasitic infections in animals).
Ma and Soave (103) introduced the Ziehl-Neelsen technique described by Henriksen and Pohlenz (65) to human medicine.
Current (38) recommended the negative staining technique of
Heine (60). A number of other techniques have been reported to be useful for demonstrating oocysts in feces (12, 13, 24,
28, 37, 72, 83, 119, 128, 203).
Detection of oocysts in feces is currently the diagnos
tic method of choice but comparative studies have failed to 33
agree on which procedure is best. Ma and Soave (103) exam ined eight methods and recommended a three step procedure using iodine wet mounts as an initial screen, Ziehl-Neelsen stain as a permanent record, and Sheather's flotation for cases in which the number of oocysts in the sample might be small. Garcia et al. (53) compared 15 methods and preferred
Ziehl-Neelsen stains of sediments of formalinized fecal sam ples which had been extracted with 10% KOH (to remove mu cus). Less comprehensive comparisons have also been made
(15, 108, 196) but no consensus has been reached on the best diagnostic method.
Prevalence The most common approach to determine the prevalence of cryptosporidiosis in human populations is to screen diarrheic fecal specimens submitted to central diagnostic laboratories. These studies have now been car ried out throughout the world and they indicate that Cryp tosporidium is common in diarrheic stool samples. Reported incidences in developed countries range from 0.35% to 5%
(14, 29, 59, 69; 70, 84, 112, 143a, 158, 185, 198, 199, 201) while incidences in third world countries vary from 4% to
10.8% (22, 68, 106, 133, 143a, 156, 192). The relative
prevalence of Cryptosporidium compared to other agents that
cause diarrhea varies from study to study but it is clear
that Cryptosporidium is of comparable prevalence to better 34
known diarrheal agents including enteropathogenic Escheri
chia coli, Campylobacter, Salmonella, Giardia, and Entamoeba
histolytica (22, 59, 69, 192).
Studies which have included non-diarrheic stool samples
have shown that the parasite is more prevalent in patients
with diarrhea than in the overall population (185, 192).
Studies which include data on other pathogens in the samples
show that the majority of specimens containing Cryp
tosporidium oocysts contain no other known pathogens (59,
106, 192). These observations strengthen the concept that
Cryptosporidium is an important cause of diarrhea in humans.
Most studies have found the parasite to be more preva
lent in children than in adults (22, 29, 112, 156, 185, 192,
199). Several studies have found a distinct seasonality to
cases of cryptosporidiosis; warm, wet weather is often as
sociated with increased incidences (69, 106, 112, 185, 192,
199).
Ep i demiclogy Cryptosporidiosis is spread through
fecal-oral contamination. Tzipori et al. (177) demonstrated
cross-transmission of Cryptosporidium from humans to mice
and lambs. Later reports documented cases of human cryp
tosporidiosis which were probably acquired from infected
animals (5, 43a, 88, 99, 140, 146). These results led to
the feeling that zoonotic infection was the most common mode
of transmission (43a, 155). 35
Soon, person to person transmission was documented
(16). Prevalence studies failed to link a large proportion of cases to animal contacts but repeatedly demonstrated clustering of cases within families and day-care centers
(22, 30, 59, 69, 169, 199). Based on these findings, it has been suggested that transmission from person to person is a more important mode of transmission than zoonotic infection
(30, 67, 82).
Infection as a result of contamination of the environ ment can also occur. An outbreak of waterborne cryp tosporidiosis probably resulting from contamination of the water supply by sewage has recently been reported (44).
Cryptosporidiosis has been documented in travelers returning from Leningrad (well known for outbreaks of waterborne giar diasis) (82, 84), Mexico (166), and the Caribbean (102).
Reported associations between cases of cryptosporidiosis and giardiasis suggest that the two parasites can be transmitted simultaneously (83, 84, 198, 199).
Treatment There is currently no treatment which will consistently cure cryptosporidiosis. In immunological
ly normal people, supportive therapy is recommended until
the disease resolves spontaneously (118). The prognosis for
immunologically suppressed people is grave unless the im
munosuppression can be reversed. Many drugs have been tried 36
in an attempt to treat cryptosporidiosis of the immunosup pressed but improvement is rarely reported (31, 118).
A few patients responded favorably to treatment with spiramycin (36, 137), raising hopes that an effective drug had been found. Most recent reports, however, suggest that the drug is inconsistent (32, 82). It must be remembered that spontaneous resolution can occasionally occur, even in the immunosuppressed (18). This observation complicates the
interpretation of chemotherapy trials in humans which are
usually uncontrolled.
Cryptosporidiosis of calves
Association of Cryptosporidium with calf scours In
the early 1970s Cryptosporidium infections were discovered
in calves dead or dying from neonatal diarrhea (calf scours)
(11, 73, 110, 121, 122, 131, 138, 153, 177). An overview of
these reports shows that the calves were typically 1-3 weeks
old, dehydrated and suffering from severe diarrhea which was
unresponsive to treatment with antibiotics. The parasites
were usually found in histological specimens taken at
necropsy. Some investigators examined the calves for other
enteric pathogens; specifically rotavirus, coronavirus and
enteropathogenic E. coli'. In some cases other pathogens
were found (129, 153) but in several they were not (11, 73,
110, 138). 37
Morin et al. (117), Moon et al. (114) and Pohlenz et al. (136) surveyed calves with diarrhea (together with healthy calves as controls) for viruses, bacteria and Cryp tosporidium. Their results showed that Cryptosporidium in fections were common in diarrheic calves but less common in healthy calves. The parasites were found both in the pres ence and in the absence of other pathogens. Snodgrass et al. (162) and Tzipori et al. (181) found Cryptosporidium to be widespread in two herds suffering scours outbreaks. In one of these herds, no other known enteropathogens were
found (181). These results suggested that Cryptosporidium
may be a cause of calf scours.
Later surveys have confirmed that Cryptosporidium in
fections are widespread among calves in general and in
calves with diarrhea in particular (6, 61, 80, 92, 124, 127,
151). Some reports suggest that all calves may encounter
the parasite at some time during their lives (1, 124, 132).
Experimental infection of calves with the parasites
resulted in clinical scours (13 6, 181, 186). The disease
was characterized by watery diarrhea, sometimes with fibrin
and mucus, which began 2-4 days after inoculation. Calves
were often depressed, emaciated and anorexic. The disease
resolved spontaneously in 8-10 days and mortality was low. 38
It was difficult for the investigators to rule out the pos
sibility that the disease was being caused by an unrecog
nized pathogen co-transmitted with the parasites. In one
study, disease appeared to be associated with contaminating
Clostridium perfringens (48). The experiments of Heine et
al. (64) using germ free calves inoculated with peracetic
acid sterilized oocysts are the strongest evidence to date
that Cryptosporidium infection alone can cause scours.
Pathology Parasites and lesions are most commonly
observed in the ileum of infected calves (11, 73, 110, 121,
129, 138, 151, 162, 186). Parasites and lesions in the
upper small intestine and large intestine are less frequent
ly reported (64, 110, 131, 136, 151, 186). The primary
microsocpic lesion is atrophy of villi (64, 73, 110, 121,
129, 138, 151, 162, 186). Villus fusions, crypt hyperpla
sia, distention of crypts with cellular debris and inflamma
tion of the lamina propria are also commonly observed (64,
73, 110, 121, 138, 151, 186). Cellular changes include re
duced epithelial cell height, sloughing of epithelial cells,
microvillus disruption and increased numbers of lysosomes in
parasitized cells (64, 73, 121, 136, 151, 162). Membrane
bound lactase activity may be decreased during the clinical
illness (186). Microscopic lesions appear to be consistent
whether or not other pathogens are present. 39
Diagnosis As with cryptosporidiosis of humans, di agnosis by demonstration of oocysts in feces is preferred.
Giemsa (136) or acid-fast stained (65) fecal smears, sucrose flotations (1, 123), and carbol-fuchsin stained native smears (60) are all effective. As in human medicine, com parisons between the various methods fail to agree on an optimum procedure (1, 132, 196).
Treatment Supportive therapy to carry calves through the clinical disease is the only approach currently • available (4). A wide range of drugs have been given to calves without success (50, 116).
Cryptosporidiosis of lambs
As researchers were becoming aware of the presence of
Cryptosporidium in calves, reports of the parasite in 1-3 week old scouring lambs began to appear (2, 11, 17, 176).
The early reports could not eliminate the possibility that other agents were responsible for the disease- Subsequent studies using germ free lambs and bacteria free inocula have strongly supported the idea that Cryptosporidium is pathogenic for lambs (161, 184).
Experimental infections showed that strains of the parasite which infected calves could also infect and produce
diarrhea in lambs (177). The intensity of Cryptosporidium-
associated diarrhea in lambs appeared to be more severe than 40
in calves and was also more severe than diarrhea due to E. coli or rotavirus in 1-2 week old lambs (177, 184).
While parasites and lesions may occur throughout the intestine they appear to be most common and severe in the ileum (11, 161, 176, 177). Pathological changes in the in testine include villus atrophy and villus fusions, disten sion and hyperplasia of crypts, inflammation of the lamina propria and reduction of epithelial cell height (10, 11, 17,
161, 177, 184). A reduction in lactase activity has also been described (184).
Cryptosporidiosis of pigs
Most reports of Cryptosporidium in pigs are based on experimental infections. These studies were initiated after it was realized that the parasite caused disease in other animals. Reports of Cryptosporidium associated with diar rhea in the field are few and do not adequately assess the potential role of other pathogens (85, 101, 152). The cur rent data suggest that cryptosporidiosis is not a signifi cant cause of scours in pigs in the field.
The results of experimental infections (including
studies of germ free animals) show that Cryptosporidium is
capable of producing diarrhea in pigs (113, 115, 177, 183,
187). The severity of the disease is reported to be greater
in artificially raised pigs than in pigs nursing a sow 41
(183). Younger animals may be more susceptible than older
ones (187). Reported prepatent and patent periods are 2-4
days and 10-17 days respectively (113, 115, 177, 183). Di
arrhea and oocyst shedding are usually closely correlated.
Intestinal lesions are similar to those found in calves and
lambs and may sometimes be severe (113, 183, 187).
In one study, respiratory and conjunctival infections
were established experimentally. Infection was associated
with microscopic lesions but not with clinical signs (63).
Cryptosporidiosis of birds
Infections with Cryptosporidium have been described in
turkeys (159), chickens (45, 52) pheasants (195), quail
(170), geese (139), peacocks (104), and pet birds (46, 54).
Many of these reports describe infection of the intestinal
tract but clinical intestinal disease has been reported only
once (159). Infections can occur throughout the lower small
intestine and large intestine but are most commonly observed
in the bursa of Fabricius (52, 77, 100, 141, 142).
Infection of the respiratory tract (especially the
trachea) is associated with disease in several host species.
Case reports typically describe flocks in respiratory dis
tress, sometimes with high mortality (104, 170, 195).
Pathological changes commonly reported are excess mucus in
the trachea, congestion and inflammation of the tracheal 42
mucosa, and epithelial hyperplasia (66, 168, 170, 195).
Parasites and lesions have also been found in the air sacks, nasal mucosa, larynx, and sinuses (56, 66, 104, 170, 195).
Infection of the conjunctiva by Cryptosporidium may be as sociated with epithelial metaplasia leading to eye abnor malities (104, 142).
Cryptosporidiosis of laboratory animals
Cryptosporidium readily infects laboratory mice. Tyz- zer (173, 174) did not observe disease in his studies of infected mice. Subsequent investigations have also failed to associate clinical disease with infection of immunologi cally normal mice (42, 47, 157). Athymic nude mice, on the other hand, develop persistent infections with diarrhea and extensive intestinal lesions (62). Mice demonstrate age- dependent resistance to infection (47, 62, 157). It is not clear if other host species also show this phenomenon.
Infection of guinea pigs has been associated with in testinal disease. Some reports describe intestinal lesions
without clinical signs (81, 190) while others describe
clinical disease and death (8). The differences may be due
to differences in strains of the parasite. 43
Infection of monkeys appears to be associated with a diarrheal syndrome not unlike that found in humans. Out breaks as well as isolated cases have been documented among macaques (35, 90, 197).
Infection of laboratory rats and rabbits has not been associated with disease (74, 146, 147, 177).
Cryptosporidiosis of other animals
Cryptosporidiosis appears to be a cause of hypertrophic gastritis in snakes (25, 167). The condition is infrequent ly reported but is characterized by severe gastric lesions leading to inappetance and eventual starvation of the animals.
Cryptosporidium has been observed in association with intestinal disease in goat kids (107, 182), red deer (179) and immunodeficient Arabian foals (55, 163). To date, the data are insufficient to assess the importance of cryp tosporidiosis in these hosts. 44
Running Title: Cryptosporidium in culture
Development of Cryptosporidium sp. in cultured cells^.
DOUGLAS B. W00DMANSEE*2 AND JOACHIM F. L. POHLENZ**
* U. S. Department of Agriculture, Agricultural Research
Service, National Animal Disease Center, Ames Iowa, *
Department of Zoology and
** Department of Veterinary Pathology, Iowa State
University, Ames, Iowa 45
1 This study was supported by the U. S. Department of
Agriculture, cooperative agreement #58-5196-2-1160.
2 Address correspondence to D. B. Woodmansee, U. S.
Department of Agriculture, National Animal Disease Center,
P. 0. Box 70, Ames, lA 50011. 46
SECTION I. DEVELOPMENT OF CRYPTOSPORIDIUM SP. IN CULTURED
CELLS
Abstract
Sporozoites of Cryptosporidium developed into immature and mature schizonts after inoculation into human rectal tumor cell cultures and into mature macro- and microgametes after inoculation into human embryonic lung cell cultures.
The relationship between parasites and the cultured cells was ultrastructurally comparable to the in vivo host- parasite relationship. The parasites were visible with
Giemsa's stain and with interference contrast microscopy.
They stained acid fast with carbol-fuchsin and reacted with anti-Cryptosporidium antibodies in an indirect im
munofluorescence test.
Introduction
In vitro growth of enteric Coccidia has been an active
area of research for several years. Doran (3) lists 28 spe
cies of Eimeria and 5 species of Isospora which have been
successfully grown in cell cultures. Of these species, only
three have been observed to undergo gametogony and only one
(Eimeria tenella) has successfully produced oocysts in vitro
when sporozoites were used as inoculum. 47
In this paper, we describe the development of Cryp tosporidium sporozoites through the asexual stages of the life cycle in a human rectal tumor (HRT) cell line and through the initiation of gametogony in a human embryonic lung (HEL) cell line.
Materials and Methods
HRT and HEL cells were routinely maintained in 75 cm^
plastic tissue culture flasks (Falcon Div., Becton, Dicker-
son and Co., Oxnard, CA) with RPMI 1640 medium (DIFCO
Laboratories, Detroit, MI) supplemented with 10%, heat inac
tivated, fetal calf serum (PCS). Cells were incubated at
370c in 5% CO2 in air and passed into new flasks regularly.
To initiate cultures for inoculation, cells were
removed from flasks by trypsinization and 1 ml of medium
containing cells was seeded into Leighton tubes containing
10.5 mm X 35 mm coverslips. Cells were allowed to grow in
the tubes for 48 hours prior to inoculation.
Feces containing Cryptosporidium oocysts were collected
from an experimentally infected calf, mixed with 5% potas
sium dichromate (1 volume feces to 2 volumes of K2Cr207
solution) and stored for up to 4 months at 4°C. Inocula for
cell cultures were prepared as shown in Fig. 1. 48
After excystation, the inocula contained fecal debris, oocysts, empty oocyst shells and free sporozoites. No sterilization of the oocysts prior to inoculation into cul tures was necessary. To inoculate cultures, the medium was
aspirated from the Leighton tubes and replaced with fresh
RPMI (no PCS, 200 units/1 penicillin, 0.2 mg/l streptomycin
sulfate, 4 g/1 amphotericin B) containing 10^ to 10^ Cryp tosporidium sporozoites.
For transmission electron microscopy, monolayers were
scraped from coverslips 1-3 days post inoculation (p.i.),
fixed in glutaraldehyde for 1-1.5 hr, processed and sec
tioned routinely, and observed with a Hitachi HS 9 electron
microscope. Samples were taken for scanning electron
microscopy 2 days p.i. Coverslips were flooded with
glutaraldehyde for 1 hr, postfixed in 1% OSO4 for 30 mi
nutes, dehydrated, critical point dried, coated with gold
paladium in a sputter coater and observed with a Joel S35 or
a Hitachi S520 scanning electron microscope.
For interference contrast microscopy, coverslips were
removed from tubes 1-6 days p.i., inverted onto a microscope
slide and observed with a Leitz Orthoplan microscope with
interference contrast optics. Cultures for staining were
removed from tubes 2 days p.i., allowed to dry, fixed in
absolute methanol, and stained with either a modified Ziehl- 49
Neelsen technique (6) or Giemsa's. Cultures were prepared for immunofluorescence microscopy 2 days p.i. using the sera
and techniques of Campbell and Current (1).
Results
Transmission electron microscopy revealed immature and
mature schizonts developing in association with HRT cells as
early as 1 day p.i. (Figs. 2-4). The morphology of the
host-parasite relationship including the parasitophorous
envelope, feeder organelle, and attachment zone appeared
similar to the morphology of these structures in vivo (8).
The morphology of the parasites themselves ranged from in
tact (Fig. 2b) to apparently degenerate (Fig. 4).
Cryptosporidia were seen in scanning electron micro
scopic (SEM) preparations made 2 days p.i. Fig. 5a shows a
mature schizont. The parasitophorous envelope has collapsed
during processing, leaving the outlines of the merozoites
visible. A similar parasite from the small intestine of a
calf is seen in Fig. 5b. Fig. 6a shows an unidentified
developmental stage in which the parasitophorous envelope is
fluted around the base of the parasite, a morphological fea
ture typical of Cryptosporidia in SEM preparations (7). A
similar stage from a calf is shown in Fig. 6b. 50
Developing parasites were visible with interference contrast microscopy 1 to 6 days p.i. They appeared as round to oval bodies, 3-5 jam in diameter, within a depression in the cells (Fig. 7). It was difficult to identify stages of the life cycle in these preparations but zoites were detect able in some specimens (Fig. 7a, 7c, 7d).
Cryptosporidia stained intensely acid-fast in cultures stained with the the modified Ziehl-Neelsen technique (Fig.
8a). The appearance of the parasites in Giemsa-stained cul tures was similar to their appearance in fecal smears (9) except that no red granules were visible (Fig. 8b). In stained preparations, it was often difficult or impossible to distinguish life-cycle stages which had developed in the culture from those introduced with the inoculum.
The parasites fluoresced brightly when treated with an indirect immunofluorescence (IIP) system directed against
Cryptosporidium (Fig. 9a). No fluorescence was observed in uninfected cultures (Fig. 9b) or infected cultures treated with non-immune sera.
Development of Cryptosporidia in HEL cells was similar to their development in HRT cells except that macro- and microgametes were observed in addition to stages of the asexual cycle (Figs. 10, 11). 51
Discussion
The results of this study show that sporozoites of
Cryptosporidium can develop at least through the asexual portion of the life cycle in HRT cells and into the sexual portion of the life cycle in HEL cells. While evidence for development beyond these stages was not found, it is pos sible that oocysts may have developed in numbers so low that they were not detected.
Current and Haynes (2) observed development through the the oocyst stage in human fetal lung cells. It may be that the choice of cell line is important in determining the ex tent and degree to which the parasites will develop in vi tro. This phenomenon has been observed in in vitro infec tions with other coccidians (4, 5, 10). To date, trial and error is the only method to determine which cell line is
optimal for a given parasite.
Transmission and scanning electron microscopy showed that the ultrastructure of the host-parasite interface, in
cluding the so-called feeder organelle, in vitro is similar
to its appearance in vivo. This indicates that in vitro
systems may provide useful models for the study of these
poorly understood structures.
The remarkably intense staining of the parasites by the
modified Ziehl-Neelsen technique may provide a basis for the 52
quantitation of in vitro infections, provided that the prob lem of differentiating developing versus introduced forms can be overcome. The reactivity of the parasites to anti-
Cryptosporidium antibodies may also provide a basis for quantification and leads to the hope that in vitro systems may eventually provide a useful source of Cryptosporidium antigens.
The failure of the parasites to produce large numbers of oocysts or to establish a persistent infection of the cultured cells precludes the use of this system as a method to produce large numbers of parasites or to maintain long term in vitro cultures for experimental or reference pur poses. However, it is still anticipated that in vitro sys tems may serve as useful tools in the study of cryp- tosporidiosis, particularly host cell-parasite interactions, parasite metabolism and the interaction of the parasites with drugs.
Literature Cited
1. Campbell, P. N. and Current, W. L. 1983. Demon
stration of serum antibodies to Cryptosporidium sp.. in nor
mal and immunodeficient humans with confirmed infections.
J. Clin. Microbiol. 18: 165-169. 53
2. Current, W. L. and Haynes, T. B. 1984. Complete development of Cryptosporidium in cell culture. Science
224: 603-605.
3. Doran, D. J. 1982. Behavior of Coccidia in vi tro. pp. 229-285. ^ Long, P. L., ed. The Biology of the
Coccidia. University Park Press, Baltimore.
4. Doran, D. J. and Augustine, P. C. 1973. Compara tive development of Eimeria tenella from sporozoites to oocysts in primary kidney cell cultures from gallinaceous
birds. J. Protozool. 20: 658-661.
5. Hammond, D. M. and Payer, R. 1968. Cultivation
of Eimeria bovis in three established cell lines and in bo
vine tracheal cell line cultures. J. Parasitol. 54: 559-
568.
6. Henriksen, S. A. and Pohlenz, J. F. L. 1981.
Staining of Cryptosporidia by a modified Ziehl-Neelsen tech
nique. Acta Vet. Scand. 22: 594-596.
7. Pearson, G. R. and Logan, E. F. 1983. Scanning
and transmission electron microscopic observations on the
host-parasite relationship in intestinal cryptosporidiosis
of neonatal calves. Res. Vet. Sci. 34: 149-154. 54
8. Pohlenz, J., Bemrick, W. J., Moon, H. W. and Che ville, N. F. 1978. Bovine cryptosporidiosis: a transmis sion and scanning electron microscopic study of some stages in the life cycle and of the host-parasite relationship.
Vet. Pathol. 15; 417-427.
9. Pohlenz, J., Moon, H. W., Cheville, N. F. and Bem rick, W. J. 1978. Cryptosporidiosis as a probable factor in neonatal diarrhea of calves. J. Vet. Med. Assoc.
172; 452-457.
10. Turner, M. B. F. and Box, E. D. 1970. Cell cul ture of Isospora from the English sparrow. Passer domesticus domesticus. J. Parasitol. 56: 1218-1223. Fig. 1. Procedures for the isolation and excystation of Cryptosporidium oocysts for inoculation into cell cultures 56
FECES IN KgCr^O? -o CENTRIFUGE 1,200 xg, 5 MIN. O . RESUSPEND IN SUCROSE SOLUTION (1.18 g/cm^)
COVER MENISCUS WITH COVERSLIP
CENTRIFUGE 1,200 xg, 5 MIN
REPLACE COVERSLIP
REPEAT 4 TIMES
POOL MATERIAL ATTACHED TO COVERSLlPS
WASH TWICE IN PBS o SUSPEND IN 0.02M CYSTEINE HYDROCHLORIDE IN 0.85% NaCL, OVERLAY WITH 50% COj IN AIR O INCUBATE, 37»C, OVERNIGHT
WASH 3 TIMES IN PBS
•o RESUSPEND IN 0.75% SODIUM TAUROCHOLATE, 0.25% TRYPSIN IN PBS -o INCUBATE 37°C. 30 MIN.
WASH IN TISSUE CULTURE MEDIUM o INOCULATE Fig. 2A-B. Mature schizonts of Cryptosporidium in HRT cells, 2 days p.i., showing parasitophorous envelope (pe), feeder organelle (fo), electron dense band (ar row) and merozoites (m). Magnification: X26,600 58 Fig. 3. Immature schizont of Cryptosporidium in HRT cells, 2 days p.i. Magnifcation: X79,800
Fig. 4. Apparently degenerate stages of Cryp tosporidium in HRT cells, 3 days p.i., showing vacuolization (V), disruption of the limiting membrane (arrows), and vesicles within the parasitophouous vacuole (v). Magnifcation; X13,300 60 Fig. 5. Scanning electron micrographs of mature schizonts showing outlines of merozoites (arrows). 5A. HRT cells 2 days p.i. 5B. From the small intestine of a calf
Fig. 6. Scanning electron micrographs of uniden tified stages of Cryptosporidium showing fluting of the parasitophorous envelope (arrows). 6A- In HRT cells 2 days p.i. 6B. From the small intestine of a calf §2 Fig- 7. Interference contrast photographs of Cryptosporidia in HRT cells. 7A-C, 3 days p.i. 7D. 4 days p.i. 64 Fig. 8. Stained Cryptosporidia in HRT cells 2 days p.i. 8A- Mature sciiizont, Ziehl-Neelsen. 8B. Unidentified stage, Giemsa
Fig- 9. HRT cells after treatment with the IIP tech nique. 9A. Infected cells. 9B. Uninfected cells
Fig. 10. Cryptosporidium macrogamete in HEL cells, 3 days p.i. Magnification: X23,500
Fig. 11. Cryptopsoridium microgamete (bottom) and unidentified stage, probably a macrogamete (top) in HEL cells, 3 days p.i. Magnification X23,500 68 69
Running title; Sporulation of Cryptosporidium
An In Vitro Study of Sporulation in Cryptosporidium sp.l
DOUGLAS B. W00DMANSEE2
Departments of Zoology and Veterinary Pathology, Iowa State
University and the U. S. Department of Agriculture,
Agricultural Research Service, National Animal Disease
Center, Ames, Iowa. 70
^ This study was supported by the U. S. Department of
Agriculture, cooperative agreement #58-519B-2-1160.
2 Address correspondence to D. B. Woodraansee, U. S.
Department of Agriculture, National Animal Disease Center,
P. 0. Box 70, Ames, lA 50010. 71
SECTION II. AjN IN VITRO STUDY OF SPORULATION IN
CRYPTOSPORIDIUM SP.
Abstract
Sporulation of Cryptosporidium oocysts was studied using an in vitro excystation procedure. No evidence was found that significant numbers of Cryptosporidium oocysts sporulate when exposed to standard in vitro sporulation con ditions, strengthening the concept that sporulation of Cryp tosporidium is completely endogenous.
Introduction
Oocysts of coccidian parasites complete their develop ment either within the host (endogenous sporulation) or out side, in the environment (exogenous sporulation). Sporula tion involves cell division within the oocyst resulting in the formation of sporozoites and, in some genera, the development of internal cyst walls to form sporocysts.
Sporulation may be induced in vitro by holding the oocysts
at room temperature with exposure to oxygen for a period of
days, usually in dilute solutions of potassium dichromate.
Coccidian oocysts are believed to be infective only if they
are sporulated (5).
Cryptosporidium sp. (Apicomplexa, Coccidiasina, Cryp-
tosporidiidae) is a pathogenic coccidian of several animal 72
species. It can infect various mucosal epithelia, but is most commonly found in the intestine (11). The parasite has an oocyst which contains four naked sporozoites and was originally described as having endogenous sporulation (10).
Subsequently, in vivo (2, 6) and in vitro (3, 4) studies confirmed that sporulation can occur while the oocyst is within the host cell. Furthermore, calf feces containing
Cryptosporidium oocysts are infective immediately after pas sage (8).
Anderson (1), however, indicated that while some oocysts were sporulated at the time of passage in feces, a substantial number were not, and that sporulation of these oocysts would occur if they were exposed to sporulating con
ditions for five to seven days. Since no objective data were presented to support these statements, it seemed worth while to examine this possibility experimentally.
The sporulation status of most coccidian oocysts can
easily be determined microscopically. While sporozoites can
be seen inside some Cryptosporidium oocysts, the small size
of the oocysts (4 to 5 jum) and the high refractiveness of
the cyst wall can make interpretation of the internal struc
ture of most oocysts difficult (Fig. 1). In this study, the
sporulation status of Cryptosporidium oocysts was analyzed
using an in vitro excystation procedure. It was reasoned
that the percentage of sporulated oocysts in a sample would 73
have an effect either on the percentage of oocysts which would excyst (if sporulated oocysts excyst more readily than unsporulated oocysts) or on the number of sporozoites released per excysting oocyst (if sporulated and unsporu- lated oocysts are equally likely to excyst).
Materials and Methods
Feces from infected calves were collected in metal pans over a 24 hour period beginning one or three days after the onset of oocyst shedding. Feces were mixed with two volumes of 2.5% potassium dichromate, and passed through a graded series of sieves (smallest sieve size 60 jam) to remove fecal debris.
The resulting suspension was placed in petri dishes
(fluid depth < 2 mm) and held at room temperature (about
23°C) without agitation. At various times after collection,
oocysts were isolated by coverslip flotation in Sheather's
sucrose solution (9) which had been diluted to a specific
gravity of 1.177 g/ml. Oocysts were washed in phosphate
buffered saline (PBS) and excysted for one hour at 37^0 in a
solution of 1.5% taurocholic acid (sodium salt, crude, Sig
ma) and 0.5% trypsin (1:250, Difco) in PBS.
After excystation, the suspension was cooled in an ice
bath, and the number of sporozoites, oocysts and empty
oocyst walls in two 1 mm^ areas of a hemacytometer were 74
determined (the sum of the numbers of oocysts and cyst walls was always greater than 200). From these data, the percent excystation [number cyst walls/(number cyst walls + number intact oocysts)] and sporozoite/cyst wall ratios were calcu lated. The assay was performed on the day of collection and
1-5, 7, 9, and 11 days after collection. The experiment was
performed twice, using oocysts from two different calves.
Results
Combined results of the experiments are shown in Fig.
2. There was no apparent correlation between either percent
excystation or sporozoite/cyst wall ratio and time in
sporulation conditions. Inspection of data from both calves
independently gave similar results.
Excystation rates below 100% and sporozoite/cyst wall
ratios below the expected value of 4 were observed. It is
likely that these low values are due to less than optimal
excystation conditions. In preliminary experiments to opti
mize excystation conditions, values approaching the
theoretical values have been observed for both parameters
(data not shown). Sporulation during the experimental peri
od should have been reflected as an upward trend in one or
both of the measured parameters, whether or not the assay
was being run at conditions optimal for excystation. 75
Discussion
The data show no indication that sporulation occurred during the 11 day observation period. It is possible that sporulation occurred between the time the feces were actual ly passed and the time the day 0 sample was assayed (about
24 hours). Most coccidia require a period longer than 24 hours to sporulate (7), but even if significant sporulation did occur, these results still do not agree with Anderson
(1), who stated that sporulation occurred in five to seven days. It is concluded that exposure of Cryptosporidium oocysts to standard sporulation conditions does not result in significant sporulation, a result consistent with the concept that sporulation of Cryptosporidium oocysts is com pletely endogenous. Any attempt to increase the number of sporozoites obtained from an in vitro excystation by first exposing the oocysts to standard conditions for sporulation of coccidia would appear to be futile. Furthermore, it is concluded that analysis of in vitro excystation is a useful tool in studies of oocyst sporulation, where microscopic determination of sporulation status is difficult.
Literature Cited
1. Anderson, B. C. 1983. Cryptosporidiosis. Lab.
Med. 14: 55-56. 76
2. Barker, M. J. and Carbonell, P. L. 1974. Cryp tosporidium agni sp. n. from lambs and Cryptosporidium bovis sp. n. from a calf with observations on the oocyst. Z.
Parasitenkd. 44; 289-298.
3. Current, W. L. and Haynes, T. B. 1984. Complete development of Cryptosporidium in cell culture. Science
224; 603-605.
4. Current, W. L. and Long, P. L. 1983. Development of human and calf Cryptosporidium in chicken embryos. J.
Inf. Dis. 148: 1108-1113.
5. Hammond, D. M. 1973. Life cycles and development of Coccidia. pp. 45-79. ^ Hammond, D. M. and Long, P. L., eds. The Coccidia: Eimeria, Isospora, Toxoplasma and Re lated Genera. University Park Press, Baltimore.
6. Iseki, M. 1979. Cryptosporidium felis sp. n.
(Protozoa: Eimeriorina) from the domestic cat. Jpn. J.
Parasitol. 28: 285-308.
7. Kheysin, Y. M. 1972. Life Cycles of Coccidia of
Domestic Animals. University Park Press, Baltimore.
8. Moon, H. W. and Bemrick, W. J. 1981. Fecal transmission of calf Cryptosporidia between calves and pigs.
Vet. Pathol. 18: 248-255.
9. Sloss, M. W. and Kemp, R. L. 1978. Veterinary
Clinical Parasitology. Iowa State University Press, Ames. 77
10. Tyzzer, E. E. 1910. An extracellular coccidium,
Cryptosporidium muris (gen. et sp. nov.) of the gastric glands of the common mouse. J. Med. Res. 23: 478-509.
11. Tzipori, S. 1983. Cryptosporidiosis in animals and humans. Microbiol. Rev. 47; 84-96. Fig. 1. Typical Cryptosporidium oocysts from calf feces. Note difficulty of interpretation of internal structure. Nomarski interference contrast, bar = 10 pm
Fig. 2. Mean excystation and sporozoite/cyst wall ratio data for oocysts from two sporulation trials using oocysts from two different calves. Failure of either parameter to change over time indicates no change in the sporulation status of the oocyst population 81
-20 w
2 3 4 5 6 7 TIME IN SPORULATION CONDITIONS (DAYS) 82
Running title: Cryptosporidium sporozoite morphology
Factors affecting motility and morphology of
Cryptosporidium sporozoites in vitro.1
DOUGLAS B. W00DMANSEE*2, EDWIN C. POWELL*,
JOACHIM F. L. POHLENZ**, and HARLEY W. MOON***
* Department of Zoology and ** Department of Veterinary
Pathology, Iowa State University, Ames, Iowa 50011, and ***
U. S. Department of Agriculture, Agricultural Research Ser
vice, National Animal Disease Center, P. 0. Box 70, Ames,
Iowa 50010. 83
^ This research was supported by USDA cooperative agreement #58-519B-2-1160. The authors wish to thank Dr.
Jerome Sacks and Mr. Danny Wolfgram for assistance with the statistical analysis and Mr. Gary Fry for technical assistance.
2 Address correspondence to D. B. Woodmansee, U. S.
Department of Agriculture, Agricultural Research Service,
National Animal Disease Center, P. 0. Box 70, Ames, lA
50011. 84
SECTION III. TAUROCHOLIC ACID AFFECTS MOTILITY AND
MORPHOLOGY OF CRYPTOSPORIDlUM SPOROZOITES IN VITRO
Abstract
In vitro motility and morphology of Cryptosporidium sporozoites was examined in the presence of various solu tions. Crude preparations of the bile salt taurocholic acid were found to affect both motility and morphology in a dose- dependent manner. These effects appeared to be due to the taurocholic acid itself and not simply due to pH variations, osmotic factors, or contaminants. Lysis of sporozoites was also observed and was found to be dependent on pH, with acidic conditions (pH < 6.2) triggering the lysis.
Introduction
Cryptosporidium sp. is a pathogenic coccidian parasite infecting intestinal, respiratory and other mucosal surfaces of several animal species (3, 5, 8). Infections are ini tiated when sporozoites excyst from oocysts onto mucosal surfaces. Sporozoites invade the apical portion of epi thelial cells and establish themselves in a position immedi
ately beneath the host cell membrane (4, 9). In addition to
its role as the infectious stage, the sporozoite has also
been implicated as the stage responsible for autoinfection,
believed to be the mechanism by which infections persist in 85
immunocompromized hosts (2, 4). Thus, the sporozoite may be a stage at which pharmacologic or immunologic attack could be of both therapeutic and prophylactic value.
Research on cryptosporidiosis would be greatly facili tated if methods for cultivation and maintenance of the or ganism in vitro could be developed. The methods for growth of Cryptosporidium in tissue culture which are currently
available (1, 10) can not be used for indefinite maintenance
or expansion of the parasite population. Knowledge of the
excystation process and the development of techniques for
the in vitro study of sporozoites are logical first steps
toward the ultimate goal of cultivation and maintenance of
Cryptosporidium in vitro. For these reasons we have been
studying in vitro excystation and sporozoite survival.
This paper describes a system for assessing sporozoite
motility and morphology. Studies using this system indicate
that Cryptosporidium sporozoites have specific environmental
requirements, the absence of which can result in morphologi
cal changes in the sporozoites.
Materials and Methods
Feces from calves experimentally infected with Cryp
tosporidium were collected, mixed with two volumes of 2.5%
potassium dichromate, passed through a graded series of
sieves and stored at 4°C for up to three months. Oocysts 86
were isolated by coverslip flotations in sucrose solution
(s.g. 1.177g/ml), sterilized by suspension in 5.25% sodium hypochlorite solution (full strength commercial bleach) for five minutes at O^C followed by three washes in sterile Dul- becco's phosphate buffered saline (DPBS), and excysted at
37°C for 30 min. The excystation solution consisted of 1.5% crude taurocholic acid (#T-0750, Sigma) and 0.5% trypsin
(1:250, Difco) in DPBS. Portions of excysted preparations were placed in plastic centrifuge tubes and centrifuged at
1000 2 for 5 min. Pellets, which contained oocysts, oocyst
shells and free sporozoites, were washed once in the test
solutions, resuspended, and the tubes were coded for blind
scoring.
Samples from each tube were placed in a phase contrast
hemacytometer and scored using Nomarski interference micros
copy. Thirty to forty five sporozoites were scored for each
sample and care was taken that sporozoites at all fluid
depths beneath the coverslip were scored.
Spcrozoites which did not move were given a motility
score of 0. Sporozoites which changed shape, flexed or
glided were assigned a motility score of 1. Sporozoites
which were shortened to less than half the length of freshly
excysted sporozoites were given a length score of 0, while
sporozoites of normal or somewhat shorter length were given
a length score of 1. Sporozoites which failed to show the 87
characteristic shape of freshly excysted sporozoites (dis tinctly crescent shaped with one end noticeably more pointed than the other) were given a shape score of 0, while sporozoites that were of the characteristic shape were given a shape score of 1. Data are expressed either as the per centage of sporozoites scoring 1 for each parameter (length, shape, motility) or as an average sporozoite score, defined as the siam of all the shape scores, length scores, and motility scores in a trial divided by the number of sporozoites scored in that trial. Data are expressed as average sporozoite scores only when trends for length, shape and motility are similar.
Osmolality and pH of some of the test solutions were determined. Average sporozoite score was plotted against osmolality and pH and the hypothesis that changes in the score were due to these factors was tested by linear regre- sion anlaysis.
In some solutions, sporozoites showed a dramatic loss of refractiveness, combined with loss of motility and ap peared thinner than freshly excysted sporozoites. This set
of changes was interpreted to represent lysis of the
sporozoites and was distinct from the length, shape and
motility changes described above. Lysed sporozoites were
not scored. 88
Results
Observations of sporozoites being prepared for inocula tion into tissue culture suggested that changes in sporozoite motility and morphology occurred when sporozoites were washed to remove excystation fluid. When the scoring system was applied, it was found that sporozoite length, shape and motility scores were significantly higher for un washed sporozoites and sporozoites washed in excystation fluid than for sporozoites washed in DPBS or 50 mM TRIS buffer supplemented with 225 mM glucose (TB). Addition of
1.5% crude taurocholic acid to DPBS or TB resulted an in crease in sporozoite scores (Table 1).
Sporozoite scores were recorded in the presence of ten
fold serial dilutions of 1.5% crude taurocholic acid in
DPBS. The results showed a correlation between sporozoite
scores and crude taurocholic acid concentration for all
three parameters (Table 2, Figs 1-5). The dilution experi
ment was repeated using a synthetic taurocholic acid prepa
ration (#T-4009, Sigma). In this experiment, the trends
between shape and length scores and taurocholic acid con
centration still held and differences between the two com
pounds were small at all dilutions (Table 2, Figs. 1,2).
Sporozoite motility scores showed differences at some dilu
tions, specifically a marked drop at the 1.5% concentration
point and a more rapid decline to baseline levels (Fig. 3). 89
Correlations between the pH and oslomolality of some test solutions and mean sporozoite score were found to be small and insignificant (-0.11 +/- 0.15 for pH and 0.1 +/-
0.007 for osmolality). Osmolarity values ranged from 278 mos to 372 mos and pH values ranged from 5.91 to 8.62.
In preliminary experiments, sporozite lysis was ob served in solutions where the pH was below 6.2, and was most readily observed in Hank's balanced salt solution (HBSS).
The hypothesis that sporozoite lysis is mediated by pH was tested by adjusting the pH of HBSS solutions with HCl, in the presence and absence of taurocholic acid. The results indicated that lysis is indeed dependent on pH (Figs. 4).
It was observed that lysis did not occur immediately but required 10 to 15 min of incubation and was arrested at 0°C.
Discussion
In this study, taurocholic acid acted to maintain motility and morphology of Cryptosporidium sporozoites in vitro. Experiments with synthetic taurocholic acid indi cated that the effects on shape and length were due to the taurocholic acid itself and not a contaminant of the crude
preparation (the type normally used for in vitro work with
coccidian sporozoites). This statement may be true for the
motility parameter as well as for the shape and length
parameters, despite the fact that motility scores declined 90
markedly in 1.5% synthetic taurocholic acid. It was not possible for us to test the synthetic and crude preparations at equimolar concentrations of taurocholic acid. The man ufacturer estimates that the crude preparation of taurocholic acid contains approximately 40% taurocholic acid by weight while the synthetic preparation contains about 98% taurocholic acid. It is possible that sporozoite motility scores would have been more similar if taurocholic acid had been present in equimolar concentrations.
The trends observed in the dilution experiments cannot be explained solely by pH or osmotic factors. The pH and osmotic pressures of the solutions studied in the dilution experiments were within the ranges tested for correlation- to average sprorozoite score. This is not to say that these factors have no effect on sporozoites at all, as evidenced
by the pH-mediated lysis observed in this study. Lysis of
sporozites at low pH suggests that excysted sporozoites may
not be able to survive passage through the stomach of an
animal.
The changes observed in this study have not previously
been reported for other coccidian sporozoites but increases
in in vitro motility of Eimeria merozoites have been ob
served in the presence of whole bile, taurocholic acid and
other bile salts (7). Other investigators have noted that
the number of Cryptosporidium sporozoites observed after 91
excystation in the presence of taurocholic acid and trypsin was greater than the number observed when the excystation was performed in buffer after hypochlorite treatment (6).
These authors hypothesized that excystation fluid enhanced sporozoite survival after excystation. Our data are consis tent with this hypothesis.
The importance of these observations to in vivo condi tions is unclear. The study suggests that the concentration of bile salts or other compounds in the in vivo environment may have important effects on sporozoites. The implications of this study for work with Cryptosporidium sporozoites in vitro are more clear. Sporozoites appear to be more deli cate and demanding of their environment than previously sup posed. Investigations of sporozoite biology should take into account the considerable effects that the in vitro en vironment (including the presence of compounds such as taurocholic acid) may have on the parasites.
Literature Cited
1. Current, W. L. and Haynes, T. B. 1984. Complete
development of Cryptosporidium in cell culture. Science
224; 603-605.
2. Current, W. L. and Long, P. L. 1983. Development
of human and calf Cryptosporidium in chicken embryos. J.
Infect. Dis. 148: 1108-1113. 92
3. Heine, J., Pohlenz, J. P. L., Moon, H. W. and Woode,
G. N. 1984. Enteric lesions and diarrhea in gnotobiotic calves monoinfected with Cryptosporidium species. J. In fect. Pis. 150; 768-775.
4. Iseki, M. 1979. Cryptosporidium felis sp. n. (Pro tozoa; Eimeriorina) from the domestic cat. Jpn. J. Parasi- tol. 28; 285-307.
5. Navin, T. R. and Juranek, D. D. 1984. Cryp- tosporidiosis: clinical, epidemiologic, and parasitologic
review. Rev. Infect. Pis. 6; 313-327.
6. Reduker, D. W. and Speer, C. A. 1985. Factors in
fluencing excystation in Cryptosporidium oocysts from cat
tle. J. Parasitol. 71: 112-115.
7. Speer, C. A., Hammond, P. M. and Kelly, G. L. 1970.
Stimulation of motility in merozoites of five Eimeria spe
cies by bile salts. J. Parasitol. 56; 927-929.
8. Tzipori, S. 1983. Cryptosporidiosis in animals and
humans. Microbiol. Rev. 47: 84-96.
9. Vetterling J. M., Takeuchi, A. and Madden, P. A.
1971. Ultrastructure of Cryptosporidium wrairi from the
guinea pig. J. Protozool. 18; 218-260. 93
10. Woodmansee, D. B. and Pohlenz, J. F. L. 1983.
Develpoment of Cryptosporidium sp. in a human rectal tumor cell line. pp. 306-319. ^ Proceedings of the 4th Interna tional Symposium on Neonatal Diarrhea. Veterinary Infectious
Disease Organization, Saskatoon. Table 1. Shape, length, and motility scores for Cryptosporidium sporozoites in
various test solutions
Total % Sporozoites Scoring 1
Sporozoites Number of Shape Length Motility
Treatment Scored Experiments + SEM + SEM + SEM
Not Washed 386 11 97 + 1 96 + 1 86 + 3
Excystation Fluid^ 200 6 95 + 1 95 + 3 83 + 3
DPBS 380 12 59 + 5 61 + 6 27 + 4
DPBS + 1.5% CTA 347 11 90 + 2 88 + 2 66 + 4
TB^ 65 2 78 + 2 67 + 4 26 + 3
TB + 1.5% CTA 63 2 97 + 3 94 + 6 74 + 14
^ Fluid consisted of 1.5% crude taurocholic acid (CTA) and 0.5% trypsin in
Dulbecco's phosphate buffered saline (DPBS).
^ 50 mMolar TRIS + 225 mM glucose in H^O, pH = 8.0. Table 2. Mean and standard error values for experiment described in Figs. 1-3, n
= 3
Taurocholic % Sporozoites Scoring
Acid Type Dilution Shape + SEM Length + SEM Motility + SEM
Crude 0 91 + 3 86 + 4 67 + 5
1 83 + 9 83 + 3 66 + 7
2 6 6 + 5 76 + 2 49 + 8
3 68 + 11 74 + 16 25 + 3
4 55 + 21 59 + 18 33 + 22
5 45 + 19 54 + 11 18 + 7
Synthetic 0 89 + 1 79 + 3 22 + 6 1 85 + 3 82 ± 3 55 + 7 2 77 + 4 69 + 5 24 + 13
3 70 + 4 69 + 9 29 + 12
4 52 + 6 44 + 11 12 + 5
5 69 + 10 62 + 9 20 + 8 Fig. 1. Sporozoite shape scores for sporozoites washed in tenfold dilutions of 1.5% crude and synthesized taurocholic acid in Dulbec- co's phosphate buffered saline (DPBS) compared to sporozoite scores in DPBS without taurocholic acid. Points represent the mean of three trials, 30-45 sporozoites were scored per trial 97
100 CRUDE TAUROCHOLATE a. SYNTHESIZED TAUROCHOLATE OPBS CO 80 •ce.
o
N 40 en
CO I I I I I 0 12 3 4 5 TAUROCHOLIC ACID DILUTION Fig. 2. Sporozoite length scores for experiment described in Fig. 1 99
= 100 h— CRUDE TAUROCHOUTE O Z SYNTHESIZED TAUROCHOUTE LU DPes
O
8 - o ^ 40H CO LU P
§5
! I I I I 0 12 3 4 5 TAUROCHOLIC ACID DILUTION Fig. 3. Sporozoite motility scores for experiment described in Fig. 1
I
I
! 101
«=« CRUDE TAUROCHOUTE O—< SYNTHESIZED TAUROCHOUTE — — — OPBS
or
CO LU 'y g
TAUROCHOLIC ACID DILUTION Fig. 4. Results of a representative experiment show ing average scores for sporozoites washed in Hank's balanced salt solution (HBSS), with and without crude taurocholic acid, at two pH levels. * = lysis observed but at least 30 intact sporozoites were found. ** = lysis so severe that 30 intact sporozoites could not be found 103
3.0-1
LU CE
HBSS+1.5 pH53 CTA pH7.0 CTA pH 5.9 pH 7.0 TREATMENT 104
Running title; Excystation of Cryptosporidium
Studies of In Vitro Excystation of Cryptosporidium sp. from
Calves.1
DOUGLAS B. W00DMANSEE2
Departments of Zoology and Veterinary Pathology, Iowa State
University and The U. S. Department of Agriculture,
Agricultural Research Service, National Animal Disease
Center, Ames, Iowa. 105
1 This study was supported by the D. S. Department of
Agriculture, cooperative agreement #58-519B-2-1160 and by a fellowship provided by the J. E. Salsbury Foundation. The author wishes to thank Ms. Deborah Hammen for technical
assistance.
2 Address correspondence to D. B. Woodmansee, U. S.
Department of Agriculture, Agricultural Research Service,
National Animal Disease Center, P. 0. Box 70, Ames, lA
50010. 106
SECTION IV. STUDIES OF IN VITRO EXCYSTATION OF
CRYPTOSPORIDIUM SP. FROM CALVES
Abstract
Studies of in vitro excystation of Cryptosporidium sp. from calves showed that sporozoite yields were optimum when oocysts were treated with bleach (sodium hypochlorite) then incubated at 37°C for 60 min in the presence of taurocholic acid solutions at pH about 7.0. Trypsin was not required for excystation and high concentrations were inhibitory.
Studies using protease inhibitors and direct assays for pro teolysis failed to implicate proteolytic enzymes as effec tors of excystation. The results suggest that Cryp tosporidium uses excystation mechanisms which are fundamen tally different from those used by Eimeria spp.
Introduction
Cryptosporidium (Apicomplexa, Coccidiasina) is a parasite of mucosal surfaces of many host species (16). It is believed to be a significant cause of diarrhea in humans
(3) and domestic animals (7). The life cycle is direct and
infection is transmitted by the ingestion of oocysts (2)..
After ingestion, coccidian oocysts release sporozoites
by a process called excystation. Excystation by members of
the coccidian genus Eimeria has been well studied and shown 107
to be a biphasic process- In the first phase, the oocyst wall is opened by either mechanical disruption or by the synergystic activities of reducing agents and C02- In the second phase, sporozoites are released from sporocysts by the activities of host digestive enzymes and surface active agents such as detergents or bile salts (11, 18).
Some coccidian species have oocysts and sporocysts which differ morphologically from Eimeria (1). The Eimeria oocyst and sporocyst walls are each interrupted by plugs known as the micropyle and Stieda body. These are removed during phases one and two, respectively. Other coccidians release sporozoites through sutures in their cyst walls. In the case of Cryptosporidium, sporocysts are absent and the suture is in the oocyst wall (2). In Sarcocystis and
Toxoplasma, the oocyst wall lacks morphological specializa tions and the suture is in the sporocyst wall (1). It has been thought that excystation mechanisms for suture bearing coccida are similar to those of Eimeria because in vitro excystation procedures which work for Eimeria also work for suture bearers.
Materials and Methods
Oocysts
Feces containing Cryptosporidium oocysts were collected
from experimentally infected calves, sieved, mixed with 108
K2Cr207/ and stored as previously described (6). Oocysts were isolated by layering 25 ml of feces over sucrose step gradients made up of 25 ml of Sheather's sucrose solution
(14) diluted to a specific gravity of 1.18 g/ml, covered by
50 ml of Sheather's diluted to 1.09 g/ml, covered by 25 ml of Sheather's at 1.22 g/ml. Gradients were centrifuged for
15 min at 900 £ in a swinging bucket rotor. The 1.09 g/ml fraction was collected and diluted in Dulbecco's phosphate buffered saline (PBS). The oocysts were pelleted by cen- trifugation at 900 £ for 10 min and stored in 2.5% K2CT2O1 at 4OC until used (up to 2 months).
Data collection and handling
After excystation, oocysts, sporozoites and cast oocyst walls were counted in a phase contrast hemacytometer (Ameri can Optical, Buffalo, NY) using Nomarski interference con trast microscopy (Leitz Orthoplan, E. Leitz and Co.,
Rockleigh, NJ). Enough 1 mm^ areas were counted for the number of oocysts and cast walls to total at least 100.
Percent excystation was calculated by dividing the number of empty cyst walls by the'sum of the cyst walls and unexcysted
oocysts. Sporozoite/cyst wall ratio was calculated by di
viding the number of sporozoites by the number of cyst
walls. The theoretical value of this ratio is 4 because 109
each oocyst contains 4 sporozoites (15). Percent theoreti cal sporozoite yield was calculated by dividing the number of sporozoites observed by four times the sum of the number of cyst walls and the number of xinexcysted oocysts. Experi ments were performed three times unless otherwise noted.
Data points on graphs represent means of three trials.
Standard errors for points on graphs ranged from +/- 2 to
+/- 15 but most points had errors within 15% of the value of the mean.
Excystation assays
Experiments to determine optimum excystation procedures used oocysts which were washed three times in cold (0°C) PBS
and once in 3 ml of cold test fluid. Pellets were resuspen-
ded in 1 ml of test fluid at room temperature (about 20°C)
and placed in a 37°C water bath. Tubes were removed to an
ice bath after the appropriate incubation time. Bleach
treatment, when used, consisted of resuspending washed
oocysts in full strength commercial bleach (5.25% sodium
hypochlorite) in an ice bath for 5 min, then washing 3 times
in cold PBS. The taurocholic acid (CTA) used was crude
taurocholic acid, sodium salt (Sigma Chem. Co., St. Louis,
MO). Trypsin was the "1:250" trypsin of Difco (Difco Labs.,
Detroit, MI). 110
To further study the effects of trypsin on excystation, oocysts were treated with bleach, resuspended in test solu tions in centrifuge tubes and placed in a 37°C water bath.
Tubes were removed to an ice bath at 5 min intervals from 0 to 30 min. All test solutions contained 0.15% CTA. To test reversibility of trypsin activity, bleach treated oocysts were incubated with 5% trypsin in PBS for 20 min at 37°C, washed 3 times in PBS, then assayed for excystation in 0.15%
CTA.
To compare the effects of bleach and cysteine/CO2 pre- treatments, one group of washed oocysts was suspended in
0.02 M cysteine HCl (Sigma) in 0.85% saline. The fluid was overlaid with a 50% mixture of CO2 and air, the tube was sealed and incubated at 370C for 16 hrs. A second group of washed oocysts was mixed with saline, overlaid with air, and incubated- At the end of the incubation, both groups were divided into two parts, one treated with bleach and washed in PBS, the other was only washed in PBS. All groups were suspended in 0.15% CTA in test tubes and excysted at 37°C.
Tubes were removed to an ice bath at intervals and scored.
To test the effects of enzyme inhibitors on excysta tion, bleach treated oocysts were resuspended to a con
centration of 1.1x10^ oocysts/ml and held on ice. Fifty jliI
of inhibitor solution or PBS and 50 pi of 0.33% CTA were
placed into test tubes in a 37°C water bath and allowed to Ill
warm for about 5 min. Ten jul of oocyst suspension were add ed to each tube at 5 min intervals for 30 min. Immediately after addition of oocysts to the last tube, all tubes were removed to an ice bath and % excystation determined. In hibitors used were phenylmethylsulfonyl fluoride (PMSF), a noncompetitive inhibitor of serine proteases; N-c<
-p-tosyl-L-arginine methyl ester (TAME), a competitive sub strate analog for serine proteases; N-e<-p-tosyl-L-lysine chloromethyl ketone (TLCK), a noncompetitive inhibitor of serine proteases with specificity for trypsin, N-tosyl-L- phenylalanine choloromethyl ketone (TPCK), a compound simi lar to TLCK but with specificity for chymotrypsin; pepsta- tin, a pepsin inhibitor; and leupeptin, a competitive in hibitor of both trypsin and papain. All inhibitors were purchased from Sigma. PMSF and TPCK were made up as stock solutions in ethanol and methanol respectively and diluted with PBS prior to use.
Azocoll assays
Bleach treated oocysts were suspended in 0.15% CTA in
0.1 M phosphate buffer (1 x 10^ oocysts/ml), pH 7.0 (PB) and
excysted for 45 min at SV^C. Azocoll (Calbiochem, San Die
go, CA), a general proteolytic substrate, was added to a
concentration of 5 mg/ml to PB, 0.1 M acetate buffer, pH
5.0, or 0.1 M Tris, pH 9.0 in 1.5 ml microfuge tubes. One 112
hundred ^1 of excysted oocysts, PB or 1 mg/ml trypsin was added to the microfuge tubes and they were incubated at 37°C for 24 hr. After incubation, tubes were centrifuged at 6630
£ for 2 min. Supernatants were collected and absorbance at
520 nm was read with a spectrophotometer.
Results
Table 1 shows results of preliminary experiments to examine the effects of trypsin and CTA on excystation of non-bleach treated oocysts. The results suggested that trypsin was unnecessary for excystation and may have been
detrimental to sporozoite survival. Reduction of CTA con
centration decreased excystation but increased sporozoite
survival. Little or no excystation was observed in the ab
sence of CTA.
Experiments to determine optimal sporozoite excystation
conditions were designed on the basis of the preliminary
experiments. Fig. 1 shows the results of time course ex
periments using non-bleach treated oocysts exposed to 0.15%
CTA. Excystation occurred rapidly between 15 and 60 min but
leveled off thereafter. Sporozoite/cyst wall ratios were
impossible to interpret at 0 and 15 min because of the small
numbers of sporozoites observed, but were close to the
theoretical value of 4 at 3 0 min. The steady decline in the
ratio after 30 min was due to disappearance of sporozoites. 113
If samples were placed in an ice bath after 30 min, excysta tion stopped and sporozoite/cyst wall ratios stabilized.
Calculation of sporozoite yields from the data from Fig. 1
shows that 1 hr of excystation gave the best sporozoite
yield (Fig. 2).
The effect of pH on excystation was examined by excyst-
ing non-bleach treated oocysts at 3T^C for 1 hr to 0.15% CTA
solutions adjusted with HCl or NaOH (Fig. 3). Higher excys
tation rates were observed at lower pH but sporozoite sur
vival was poor at pH £ 6.5. pH vales of approximately 7.0
gave optimum sporozoite yields.
Non-bleach treated oocysts exposed to two-fold serial
dilutions of 1.5% CTA were incubated at 370c for 1 hr to
determine the effects of taurocholic acid concentration
(Fig. 4). Substantial excystation occurred over a wide
range of concentrations but values tended to decline as the
concentration was reduced. Sporozoite/cyst wall ratios
similarly declined as CTA concentrations were reduced.
The following experiments were performed to examine
effects of various pretreatments on excystation rather than
to optimize sporozoite yields; thus, sporozoite survival
data were not collected. Fig. 5 shows the results of side-
by-side comparisons of bleached and unbleached oocysts in
the presence and absence of taurocholic acid. Bleach treat
ment enabled excystation to occur in the absence of CTA. 114
Highest levels of excystation were observed when bleach treatment was combined with taurocholic acid exposure.
Fig. 6 shows the effect on excystation of exposure of oocysts to cysteine HCl and CO2. Data are expressed as change in % excystation (excystation value at time 0 sub tracted from excystation values for each subsequent time) because spontaneous excystation of 5-10% was observed after incubation for 16 hr at 37®C either in the presence or ab sence of cysteine/C02. Treatment of oocysts with cysteine/
CO2 increased excystation rates upon exposure to 0.15% CTA compared to controls which were incubated in saline. The combination of cysteine/CO2 and bleach treatments did not increase excystation rates over bleach treatment alone. The
controls in this experiment were expected to behave similar
ly to non-bleach treated oocysts but excystation after 30
min exposure to CTA was low. Presumedly, exposure of
oocysts to 37°C for 16 hr adversely affected their ability
to respond to taurocholic acid.
Figs. 7-9 show results of experiments to further ex
amine the potential role of trypsin in excystaion. Addition
of 10-50 mg/ml of trypsin to 0.15% CTA reduced excystation
in a dose-dependent manner (Fig. 7). Inhibition by trypsin
was found to be reversible (Fig. 8) and could not be
mimicked by bovine serum albumin (Fig. 9). 115
PMSF, TAME, TPCK, TLCK, leupeptin, and pepstatin could not be shown to inhibit excystation (Figs. 10-15), but low concentrations of ethanol and methanol were inhibitory
(Figs. 10, 13). Attempts to detect protease activity with
Azocoll were unsuccessful (Table 2).
Discussion
From the results of these experiments it is possible to suggest efficient conditions for in vitro excystation of
Cryptosporidium sporozoites. Taurocholic acid is required unless the oocysts are bleach-treated, but concentrations can be varied over a wide range with little effect on sporozoite recovery. Best excystation is achieved when bleach pretreatment is combined with taurocholic acid expo sure. Sixty minute incubations at 37^0, pH 7.0 gave the
best results in this study.
Sporozoites were short-lived at 37°C and removal of the
sporozoites to an ice bath after excystation stabilized
sporozoite numbers. This result demonstrates that excysta
tion and sporozoite loss are temperature dependent and that
holding the samples in an ice bath until they were scored
did not affect the outcome of the kinetic experiments. In. a
previous investigation, sporozoite loss after excystation
was observed to a far lesser extent (12). 116
Examination of the kinetics of excystation consistently revealed a lag time of 10-15 min before excystation began.
Excystation occurred rapidly from 15-60 min and leveled off by 90 min. In the only experiment in which lag time was
reduced (Fig. 6), the oocysts were incubated at 37°C over
night prior to the experiment which seemed to change the
oocyst's reactivity to taurocholic acid. The consistent
appearance of the lag under standard conditions suggests
that it is a necessary consequence of the excystation
mechanism and may be a clue to the nature of the mechanism.
These experiments reinforce the role of bile salts as a
trigger for excystation of Cryptosporidium. The conclusion
of Payer and Leek (5) that excystation can occur without
host stimuli was not strongly supported, except in the case
of bleach treated oocysts. My results are consistent with
and extend those of Reduker and Speer (12).
Reduker and Speer (12) hypothesized that bleach treat
ment removed a portion of the oocyst wall normally removed
by bile and/or trypsin, but this speculation was not sup
ported by morphologic evidence published subsequently by the
same group (13). The observation of multiplicative effects
between bleach and taurocholic acid found in the current
study suggests that the two stimuli work via different
mechanisms. 117
Failure of bleach and cysteine/C02 pretreatments to show additive effects suggests that they operate via the same mechanism. Cysteine/CO2 increases the number of sulfhydryl groups on the exterior of Eimeria oocysts (10) and is thought to mimic stomach passage in vivo (9). Bleach treatment may also mimic some process that occurs in the stomach.
The observations that trypsin is unnecessary for excys tation and that high concentrations are inhibitory were un expected. Previous publications describing excystation of
Cryptosporidium consistently include trypsin as part of the excystation fluid (5, 12, 13, 17, 19). Experiments using protease inhibitors and the artificial protease substrate
Azocoll failed to implicate proteolytic enzymes as mechanisms for excystation- Since host-derived proteolytic enzymes are believed essential to the excystation of Eimeria
(11), the excystation mechanism used by Cryptosporidium would appear to be fundamentally different.
Payer and Leek (4) observed excystation of Sarcocystis fusiformis sporocysts in the absence of trypsin provided that the sporocysts were treated with cysteine/C02• Their results, combined with mine, suggest that all suture bearing coccidia may use an alternate excystation mechanism.
The existence of parasite-derived enzymatic pathways for excystation has been proposed for Cryptosporidium (5) 118
and other coccidians (8, 9). The evidence suggests that enzymes are involved in excystation of Cryptosporidium since the process is temperature dependent and responsive to changes in pH. The excystation of bleach treated oocysts requires only a rise in temperature and therefore must be effected by mechanisms within the oocyst. Reduker et al.
(13) showed that the morphological changes during excysta tion appear first on the internal aspects of the excystation suture, further supporting the idea that excystation is ef fected from the inside.
The current experiments raised important questions about the mechanism of excystation in Cryptosporidium and perhaps the other suture bearing coccidia. The data suggest that oxidation/reduction reactions and bile salts can serve
as triggers for excystation- It appears that these triggers
do not just pave the way for the activity of host pro
teolytic enzymes. They may, in fact, activate an enzymatic
system inside the oocyst which, in turn, effects
excystation.
Literature Cited
1. Chobotar, B. and Scholtyseck, E. 1983. Ultra-
structure. pp. 101-106. In Long, P. L., ed. The Biology
of the Coccidia. University Park Press, Baltimore. 119
2. Current, W. L. and Reese, N. C. 1986. A compari son of endogenous development of three isolates of Cryp tosporidium in suckling mice. J. Protozoal. 33; 98-108.
3. Current, W. L., Reese, N. C., Ernst, J. V., Bailey,
W. S., Heyman, H. B. and Weinstein, W. M. 1933. Human cryptosporidiosis in immunocompetent and immunodeficient persons. N. Engl. J. Med. 308: 1252-1257.
4. Payer, R. and Leek, R. G. 1973. Excystation of
Sarcocystis fusiformis sporocysts from dogs. Proc. Helm.
Soc. Wash. 40: 294-296.
5. Payer, R. and Leek, R. G. 1984. The effects of
reducing conditions, medium, pH, temperature, and time on in
vitro excystation of Cryptosporidium. J. Protozool. 31:
567-569.
6. Heine, J., Moon, H. W. and Woodmansee, D. B. 1984.
Persistent Cryptosporidium infection in congenitally athymic
(nude) mice. Infec. Immun. 43; 856-859.
7. Heine, J., Pohlenz, J. P. L., Moon, H. W. and
Woode ; G. N. 1984, Enteric lesions and diarrhea in
gnotobiotic calves monoinfected with Cryptosporidium spe
cies. J. Inf. Pis. 150: 768-775.
8. Hibbert, L. E. and D. M. Hammond. 1968. Effects
of temperature on in vitro excystation of various Eimeria
species. Exp. Parasitol. 23: 161-179. 120
9. Jackson, A. R. B. 1962. Excystation of Eimeria arlonqi (Marotel, 1905): stimuli from the host sheep. Na ture (London) 194: 847-849.
10. Jolley, W. R., Burton, S. D., Nyberg, P. A. and
Jensen, J. B. 1976. Formation of sulfhydryl groups in the walls of Eimeria stiedai and E. tenella oocysts subjected to
in vitro excystation. J. Parasitol. 62; 119-202.
11. Ryley, J. F. 1973. Cytochemistry, physiology,
and biochemistry, pp. 145-181. In Hammond, D. M. and Long,
P. L., eds. The Coccidia; Eimeria, Isospora, Toxoplasma.
and Related Genera. University Park Press, Baltimore.
12. Reduker, D. W. and Speer, C. A. 1985. Factors
influencing excystation in Cryptosporidium oocysts from cat
tle. J. Parasitol. 71: 112-115.
13. Reduker, D. W., Speer, C. A. and J. A. Blixt.
1985- Ultrastructural changes in the oocyst wall during
excystation of Cryptosporidium parvum (Apicomplexa; Eucoc-
cidiorida). Can. J. Zool. 63: 1892-1896.
14. Sloss; M- W. and Kemp. R. L. 1978. Veterinary
Clinical Parasitology. Iowa State University Press, Ames,
lA.
15. Tyzzer, E. E. 1910. An extracellular coccidium,
Cryptosporidium muris (gen, et sp. nov.) of the gastric
glands of the common mouse. J. Med. Res. 23: 478-509. 121
16. Tzipori, S. 1983. Cryptosporidiosis in animals and humans. Microbiol. Rev. 47; 84-96.
17. Upton, S. J. and Current, W. L. 1985. The spe cies of Cryptosporidium (Apicomplexa: Cryptosporidiidae) infecting mammals. J. Parasitol. 71: 625-629.
18. Wang, C. C. 1983. Biochemistry and physiology of
Coccidia. pp. 167-228. ^ Long, P. L., ed. The Biology of the Coccidia. University Park Press, Baltimore.
19. Woodmansee, D. B. and Pohlenz, J. F. L. 1983.
Development of Cryptosporidum sp. in a human rectal tumor cell line. pp. 306-317. ^ Proceedings of the Fourth
International Symposium on Neonatal Diarrhea. Veterinary
Infectious Disease Organization, Saskatoon. Table 1. Excystation of non-bleach-treated oocysts in different excystation solu
tions
Mean
Sporozoite
Base Mean % Cyst Wall
Solution [T.A. [TRYP]^ Time^ Excystation Ratio N
DPBS 1.5 0.5 30 56 1.54 4
DPBS 1.5 0 30 52 2.62 4
DPBS 0.15 0 30 38 4.11 3
DPBS 0 0 30 0 ——— 3
HgO 0 0 60 2 0.33 3
^ Concentration (% w/v) of crude taurocholic acid.
^ Concentration (% w/v) of trypsin.
In minutes. Table 2. Results of incubation of Azocoll with 0.15% CTA and 0.15% CTA containing
1 X 10^ excysted Cryptosporidium oocysts. Differences between oocyst
and no oocyst groups are not statistically significant (P <0.05)
Mean
Buffer pH Oocysts + SEM N
Phosphate 7.0 No 0.256 + 0.027 8
Phosphate 7.0 Yes 0.209 + 0.022 8
Tris 9.0 No 0.502 + 0.052 5
Tris 9.0 Yes 0.498 + 0.021 10
Acetate 5.0 No 0.138 + 0.017 8
Acetate 5.0 Yes 0.131 + 0.018 8 Fig. 1. Time course for excystation of non-bleach treated oocysts. Dashed lines represent values for tubes placed in ice at 30 min. Excystation conditions: 0.15% CTA in PBS, 370c
Fig. 2. Sporozoite yield calculated from data in Fig. 1 % THEORETICAL SPOROZOITE YIELD % EXCYSTATIÛM oôë^êSSSSSS o
CJI
o
—I SPOROZOITE/CYST WALL RATIO m 3 _ ro o 5 o 3
s-
o Fig. 3. Effects of pH on excystation of non-bleach treated oocysts. Excystation conditions: 0.15% CTA, 370c, 60 min
Fig. 4. Effects of CTA concentration on excystation of non-bleach treated oocysts. Dilutions are two-fold dilutions of 0.15% CTA in PBS. Excystation conditions: 37°C, 60 min., pH 7.0 % EXCYSTATION % EXCYSTATION ooSSêSSSSSS
cr> - H to SPOROZOITE/CYST WALL RATIO SPCROZOITE/CYST WALL RATIO O ro CM O -L.
ro Fig. 5. Effects of bleach treatment on excystation in the presence and absence of 0.15% CTA. Excystation conditions; 37°C, pH 7.0
Fig. 6. Effect of cysteine HCl and CO2 pretreatment compared to bleach treatment. Excystation conditions: 0.15% CTA, 370C, pH 7.0 129
100 T A NO TREATMENT BLEACH TREATED XaUROCHOLIC ACID EXPOSED BLEACH TREATED + TAUROCHOIIC ACID EXPOSED
60- co ^40 4 I t t
20 -
60 90 120 TlME(min)
100 ùr-ù>. CYSTIE* HQf BLEACH 0-K3 BLEACH TREATED CTSTIENE HCt TREiSTED 80 A—A NO TREATMENT
i 60 1 ^ 40
20
0 10
TlMElmin) Fig. 7. Effect of trypsin on excystation of bleach treated oocysts. Excystation conditions as in Fig. 6
Fig. 8. Reversibility of trypsin effects. Treatment conditions: 5% trypsin in PBS, 21'^C, 20 min, washed 3X in PBS. Excystation conditions as in Fig. 6 131
MO TRYPSIN
en ^ 40 /15% TRYPSIN. LU
TIME (min)
80 NO PRETREATMENT
60 TRYPSIN PRETREATMENT
0
TIME (min) Fig. 9. Effects of BSA on excystation of bleach treated oocysts- Excystation conditions as in Fig. 6 % EXCYSTATION o o o o 8 I I I I I I I tm K K s if î g ï. » g g §
H U> W Figs. 10-15. Effects of protease inhibitors on ex cystation of bleach treated oocysts. \ Excystation conditions as in Fig. 6. Fig. 10. PMSF in ethanol and ethanol alone
Fig. 11. TAME, experiment similar to Fig. 10 135
100 0PNSF,0 ETHANOL /ml ùr^ 8.9 ni ETHANOL/ml O—• 8.9wl ETHANOL + 89 jig PNSF/ ml
^ 40 -
T r 10 15 20 TIME (min)
100
a—a 9IO,ig TWE/ml
60- co Pu 40 -
20-
15 20 25 30 TIME (mini Fig. 12. TLCK, experiment similar to Fig. 10
Fig. 13. TPCK in methanol and methanol alone, ex periment similar to Fig. 10 137
100 T O/ig TLCX/ml 455/iq TUCK/ml D—• 910/uq TICK/ml 80-
O B 60 CO >- X LU 40- é / 20- / /-
10 15 20 25 30 TIME (min]
100 0TPCX,0 METHANOL/ml 10.2^1 METHANOL/ml 380 lUj TPCK + 10.2 ^1 METHANOL / ml
O i5 60- CO X 40 -
TlME(min) Fig. 14. Pepstatin, experiment similar to Fig. 10
Fig. 15. Leupeptin, experiment similar to Fig. 10 % EXCYSTATION % EXCYSTATION o È o o S j «J u
en • II
o -| g H 9 U) m VD Ul
8^ 140
Running title: Isolation of Cryptosporidium
Techniques for the Isolation of Cryptosporidium Oocysts and
Sporozoites from Calf Feces.^
DOUGLAS B. W00DMANSEE*2 and THOMAS A. CASEY**
*Departments of Zoology and Veterinary Pathology, Iowa State
University and **The National Animal Disease Center, Ames,
Iowa, 50011 141
1 This study was supported by The United Stated Depart ment of Agriculture, cooperative agreement #58-5195-2-1160 and by a fellowship from the Salsbury Foundation. The au thors wish to thank Ms. Deborah Hammen and Mr. Gary Fry for technical assistance. No endorsements are herein implied.
2 Address correspondence to D. B. Woodmansee, U. S.
Department af Agriculture, National Animal Disease Center,
P. 0. Box 70, Ames, lA 50011. 142
SECTION V. TECHNIQUES FOR THE RECOVERY OF CRYPTOSPORIDIUM
OOCYSTS AND SPOROZOITES FROM CALF FECES
Abstract
Cryptosporidium oocysts were isolated from calf feces using sucrose step gradients. The gradients yielded oocysts in two grades of purity, Grade A (higher purity) and Grade B
(lower purity). Oocysts could be further purifed by extrac tion with chloroform or 5% SDS followed by washing over
Nuclepore filters. The resulting suspensions could be treated with bleach and taurocholic acid to excyst sporozoites. Purified sporozoites could be recovered by filtration.
Introduction
Studies of the biochemistry and molecular biology of
Cryptosporidium (Apicomplexa, Coccidiasina) have been ham pered by difficulties in obtaining adequate numbers of clean oocysts and sporozoites» A number of techniques for isola tion of Cryptosporidium oocysts from feces have been re
ported (3, 5, 6, 7). No procedures for producing purified
sporozoites have yet been reported. In our hands, percoll
gradients, coverslip flotations, glass bead filtrations and
DEAE cellulose chromatography were inadequate for isolation 143
of oocysts from feces or sporozoites from excystation mix tures despite the fact that all of these methods have been reported useful for isolation of various life cycle stages
of other coccidians. Therefore, we developed new techniques
which may be of wide spread utility.
Materials and Methods
Animals
Calves, purchased from local farms or born at the cen
ter, were immediately housed in isolation in calf crates.
They were inoculated with 10^ Grade B oocysts (see below)
mixed with the feed as soon as they were eating well (usual
ly less than 24 hrs). Feed was a commercial milk replacer
(Land '0 Lakes) used according to manufacturer's instruc
tions. Calves were checked daily for oocyst shedding by
carbol-fuchsin stained fecal smears (1).
When calves developed diarrhea and began to shed heavi
ly, they were taken off feed and given a commercial oral
rehydration solution (Life Guard, Norden). Heavy shedding,
which typically began three to four days post inoculation
(p.i.), was defined as ++ or higher on a + to ++++ scale
(2). Feces were allowed to fall into metal pans containing
about 500 ml of a 2.5% potassium dichromate solution and
were collected daily for four to six days depending on the 144
duration of heavy shedding. After the feces were collected, the calves were returned to noinaal feed and allowed to re cover spontaneously.
Preparation of raw pools
Feces and dichromate from the pans were stored at 4^0 until collection was complete. The material was then passed through standard testing sieves as previously described (2).
Sieved fecal suspensions were centrifuged at 4600 £ to pel let the solid material. Pellets were resuspended in ap proximately equal volumes of 2.5% potassium dichromate, pooled, and stored at 4^0 for up to three months.
Isolation of Grade A and Grade B oocysts
Sucrose step gradients were formed by placing 25 ml of
Sheather's sucrose solution (4) diluted to a specific gravi ty of 1.18 g/ml in the bottom of a 200 ml glass centrifuge bottle. This was overlaid by 50 ml of Sheather's diluted to
1-09 g/ml, 25 ml of Sheather's at 1.02 g/ml and 25 ml of the raw pool. Gradients were centrifuged at 900 £ for 15 min in
a swinging bucket rotor.
After centrifugation, the 1.09 g/ml fraction was col
lected and diluted with 3 volumes of Dulbecco's phosphate
buffered saline (PBS). The bands at the 1.18/1.09 and 1.09/
1.02 interfaces were collected into a separate container and 145
also diluted. The remainder of the gradient was discarded.
Solid material was collected from both fractions by cen- trifligation at 900 £ for 10 min and resuspended in 2.5%
potassium dichromate. Oocysts obtained from the 1.09 g/ml
fraction were called Grade A while those from the bands were called Grade B,
Preparation of purified oocysts
Lipids were removed from oocyst suspensions by mixing
equal volumes of Grade A or Grade B material and chloroform,
and immediately separating the phases by a brief centrifuga-
tion. The water phase was collected, leaving the layer of
debris at the interface of the phases. This was repeated
until the water phase was almost transparent. In an alter
nate procedure, Grade A material was extracted by two washes
in 5% sodium dodecyl sulfate (SDS) in 0.05M Tris buffer, pH
9.0 followed by one wash in PBS.
After extraction, oocysts were placed in a stirred cell
(Model 52, Amicon Corporation, Danvers, MA) fitted with a
Nuclepore membrane with a 3 ;ain pore size (#111012, Nuclepore
Corporation, Pleasanton, CA). The stirred cell was connect
ed to a reservoir (#XX6700P05, Millipore Corporation, Bed
ford, MA) containing 2 liters of sterile PBS. PBS was
flushed through the cell under pressure (5-10 psi) with
vigorous stirring until all the PBS except 10 ml had passed 146
through. Oocysts were retained by the membrane while small debris were washed through the pores. The last 10 ml were left stirring in the cell for 30 min to remove oocysts from the surface of the membrane. The purified oocysts were re covered from the stirred cell, placed in sterile injection vials and stored at 4°C.
Preparation of sporozoites
Purifed oocysts were mixed with full strength commer cial bleach (5.25% sodium hypochlorite) for 5 min at O^C then washed three times in PBS. Washed oocysts were sus pended in warm, 0.15% taurocholic acid (#T-0750, Sigma
Chemical Company, St. Louis, MO) in PBS and placed in the stirred cell in a 37^0 incubator and stirred slowly.
Sporozoites were collected after 30 min by carefully drawing fluid through the membrane with a syringe. The filtrate was placed in a centrifuge tube in an ice bath and fresh taurocholic acid solution was added to the cell. A second
group of sporozoites was collected after 30 min and pooled
with the first.
Results and Discussion
Our laboratory is concerned primarily with cryp-
tosporidiosis of cattle. Cryptosporidium infections of 147
calves follow a predictable clinical course and produce pro
digious numbers of oocysts (lOlO-loll oocysts per animal),
thus they were an obvious source of oocysts and sporozoites.
Our early efforts to isolate oocysts were hampered by
inconsistent behavior of feces-dichromate mixtures. The
practice of placing the calves on oral rehydration solu
tions, which have less solid matter than milk replacer,
eliminates some of the variability (W. L. Current, Lilly
Research Laboratories, Greenfield, IN, pers. comm.). Pel
leting and resuspending the sieved suspensions also led to
more consistent results. Oocyst numbers in the raw pools
were difficult to determine because of the large amount of
debris, but in one typical pool, the oocyst concentration
was estimated to be 8x10^ oocysts/ml. Four to five liters
of raw pool per calf were obtained.
Examination of the various fractions from the sucrose
gradients showed that oocysts could be found in all of them,
but large numbers of oocysts appeared in the 1.09 g/ml frac
tion and in the two bands. In one typical isolation, 42% of
the oocysts loaded onto the gradient were recovered as Grade
A while 22% were recovered as Grade B. Yield and purity
varied from raw pool to raw pool.
Grade A material had a better ratio of oocysts to de
bris than Grade B. In the remainder of the gradient, the
ratio of oocysts to debris was so poor that the oocysts were 148
not used. Grade B oocysts were clean enough to be accurate ly counted in a hemacytometer and are routinely used in this laboratory for inoculation of experimental animals. There were no obvious morphological differences between Grade A oocysts and Grade B oocysts.
The filtration of small debris through Nuclepore fil ters was found to be an excellent method of purification of oocysts provided that the material was extracted with chlo roform or SDS beforehand. Insufficient extraction resulted in rapid plugging of the membranes by an amorphous material, presumedly lipid. Chloroform extraction usually gave lower yields than SDS extraction but oocyst purity after filtra tion was excellent. SDS extraction gave consistently high yields, but in certain pools large debris contaminating the oocysts were retained in the final preparation. The number of oocysts which could be filter purified at one time was limited to 1-2x10® because the oocysts themselves can oc clude the holes in the membrane, resulting in unacceptably low flow rates. Recovery of purifed oocysts from the stirred cell was good provided that plugging did not occur and that the oocysts were vigorously stirred to remove them from the surface of the membrane.
The number of sporozoites recovered was limited primarily by the number of purified oocysts available.
Plugging of membranes during sporozoite isolations was not 149
observed. Sporozoite recoveries ranged from one sporozoite per oocyst to almost four sporozoites per oocyst. The primary contaminant of these preparations was residual bodies, but contamination by oocysts and cast oocyst shells also occurred. In the worst case of contamination observed, sporozoite numbers were 100 times higher than cast shell numbers and 10,000 times higher than oocyst numbers. On occasion, we obtained preparations with no detectable oocyst or cast shell contamination.
The procedures described are now in routine use in this laboratory. They have proven to be reliable methods for the isolation of oocysts and sporozoites. Filtration of ex tracted oocysts is currently the limiting step. We are at tempting to modify the procedures to increase yields from this step.
Literature Cited
1. Heine, J. 1982. Eine einfache Nachweismethode fur
Kryptosporidien im Kot. Zentralbl. Veterinaermed. Reihe B
29: 324-327.
2. Heine, J., Moon, H. W. & Woodmansee, D. B. 1984.
Persistent Cryptosporidium infection in congenitally athymic
(nude) mice. Infect. Immun. 43; 856-859. 150
3. Heyman, M. B., Shigekuni, L. K. & Amman, A. J-
1986. Separation of Cryptosporidium oocysts from fecal de bris by density gradient centrifugation and glass bead columns. J. Clin. Microbiol. 23; 789-791.
4. Sloss, M. W. and Kemp, R. L. 1978. Veterinary
Clinical Parasitology. Iowa State University Press, Ames,
lA.
5. Ungar, B. L. P., Soave, R., Payer, R. & Nash, T. E.
1986. Enzyme immunoassay detection of immunoglobulin M and
G antibodies to Cryptosporidium in immunocompetent and im-
munodeficient persons. J. Infect. Pis. 153: 570-578.
6. Waldman, E., Tzipori, S. & Forsyth, J. R. L. 1986.
Separation of Cryptosporidium species oocysts from feces by
using Percoll discontinuous density gradient. J. Clin.
Microbiol. 23; 199-200.
7. Woodmansee, D. B. & Pohlenz J. F. L. 1983.
Development of Cryptosporidium sp. in a human rectal tumor
cell line. pp. 306-317. ^ Proceedings of the Fourth
International Symposium on Neonatal Diarrhea. Veterinary
Infectious Disease Organization, Saskatoon. 151
GENERAL SUMMARY
Review of the published literature on Cryptosporidium shows that most of the available information is observation al. Experimental work has been hampered in part by a lack of in vitro techniques. This thesis reports efforts to develop new techniques and to apply techniques used to study other coccidian parasites to Cryptosporidium. Each of the papers presented here addresses a topic related to the han dling of Cryptosporidium in vitro.
In the work reported in Section I, standard techniques were used in an attempt to grow the parasites in cell cul ture. While development of the parasites through most of the life cycle was observed, the system was found to be in
sufficient in that productive infections were not es
tablished. These initial studies pointed out a need for
more information about the behavior of the parasites during
and immediately after excystation.
Section II answers a question raised by a paper
published by Anderson (3), in which he stated that substan
tial sporulation occurred after oocysts are passed in calf
feces. The question is important because if sporulation
does occur, then sporozoite yields from in vitro excysta-
tions should increase if one passes the oocysts through the
sporulation procedure. My experiments showed no increase in
sporozoite yields after running a sporulation procedure. 152
This result is consistent with the long standing belief that sporulation of Cryptosporidium oocysts is complete when the oocysts are shed.
Section III reports findings related to survival of sporozoites after excystation. Taurocholic acid was found to help in the maintenance of motility and morphology of sporozoites. Neutral pH and low temperatures also improved sporozoite survival. These findings should alert research ers that Cryptosporidium sporozoites may be more delicate than sporozoites of other coccidians and help researchers to develop protocols which maximize sporozoite survival.
Section IV reports the effects of various factors on in vitro excystation. In addition to establishing optimum (in my hands) excystation conditions, the data suggest that the mechanisms of excystation may be different for Cryp tosporidium than they are for Eimeria. Reported procedures
for excystation of Cryptosporidium are essentially copies of
procedures for excystation of Eimeria. Review of the
literature gives one reason to believe that other coccidian
parasites may also use alternate excystation mechanisms.
Elucidation of these mechanisms may lead to more efficient
approaches to in vitro excystation of these species and per
haps to approaches to block excystation in vivo.
Section V reports new methods of isolation of Cryp
tosporidium oocysts from feces and sporozoites from oocysts. 153
We are currently using oocysts and sporozoites isolated by the new procedures in studies of the biochemistry and
molecular biology of sporozoites. It is hoped that these
procedures will come into widespread use and that they will
contribute substantially to our understanding of the biology
of Cryptosporidium. 154
LITERATURE CITED
1. Anderson, B. C. 1981. Patterns of shedding of cryptosporidial oocysts in Idaho calves. J. Vet. Med.
Assoc. 178: 982-984.
2. Anderson, B. C. 1982. Cryptosporidiosis in Idaho lambs: natural and experimental infections. J. Vet.
Med. Assoc. 181: 151-155.
3. Anderson, B. C. 1983. Cryptosporidiosis. Lab.
Med. 14: 55-56.
4. Anderson, B. C. and Bulgin M. S. 1981. Enteritis caused by Cryptosporidium in calves. Vet. Med. Small Anim.
Clin. 76: 865-868.
5. Anderson, B. C., Donndelinger, T., Wilkins R. M. and Smith, J. 1982. Cryptosporidiosis in a veterinary stu dent. J. Vet. Med. Assoc. 180: 408-409.
6. Anderson, B. C. and Hall, R. F. 1982. Cryp tosporidial infection in Idaho dairy calves. J. Vet.
Med. Assoc. 181: 484-485.
7. Andreani, T., Modigliani, R., Le Charpentier, Y.,
Galian, A., Brouet, J. C., Liance, M., Lachance, J. R.,
Messing, B. and Vernisse, B. 1983. Acquired im
munodeficiency with intestinal cryptosporidiosis: possible
transmission by Haitian whole blood. Lancet i: 1187-1191.
8. Angus, K. W., Hutchison, G. and Munro, H. M. C.
1985. Infectivity of a strain of Cryptosporidium found in 155
the guinea-pig (Cavia porcellus) for guinea-pigs, mice and
lambs, J. Comp. Pathol. 95: 151-165.
9. Angus, K. W., Sherwood, D., Hutchison, G. and Camp
bell, I. 1982, Evaluation of the effect of two aldehyde-
based disinfectants on the infectivity of faecal Cryp
tosporidia for mice. Res. Vet, Sci, 33: 379-381.
10. Angus, K. W,, Tzipori, S. and Gray, E, W, 1982,
Intestinal lesions in specific-pathogen-free lambs associ
ated with a Cryptosporidium from calves with diarrhea. Vet.
Pathol. 19: 67-78.
11. Barker, I. K. and Carbonell, P, L, 1974. Cryp
tosporidium aqni sp. n, from lambs, and Cryptosporidium
bovis sp, n. from a calf with observations on the oocyst.
Parasitenkd. 44: 289-298.
12. Baxby, D, and Blundell, N. 1983. Sensitive, rap
id, simple methods for detecting Cryptosporidium in faeces.
Lancet ii: 1149.
13. Baxby, D,, Blundell, N, and Hart, C. A, 1984,
The development and performance of a simple, sensitive meth
od for the detection of Cryptosporidium oocysts in faeces.
J. H^, 92: 317-323.
14. Baxby, D. and Hart, C. A. 1984. Cryp-
tosporidiosis, Br, Med, J. 289: 1148. 156
15. Baxby, D., Hart, C. A, and Blundell, N. 1985.
Shedding of oocysts by immunocompetent individuals with cryptosporidiosis. J. Hyg. 95; 703-709.
16. Baxby, D., Hart, C. A. and Taylor, C. 1983. Hu man cryptosporidiosis; a possible case of hospital cross infection. Br. Med. J. 287; 1760-1761.
17. Berg, I. E., Peterson, A. C. and Freeman, T. P.
1978. Ovine cryptosporidiosis. J. Vet. Med. Assoc.
173: 1586-1587.
18. Berkowitz, C. D. and Seidel, J. S. 1985. Spon taneous resolution of cryptosporidiosis in a child with ac quired immunodeficiency syndrome. J. Pis. Child. 139;
967.
19. Bird, E.G. and Smith, M. D. 1980. Cryp tosporidiosis in man: parasite life cycle and fine struc tural pathology. J. Pathol. 132: 217-233.
20. Blumberg, R. S., Kelsey, P., Perrone, T., Dicker- son, R., Laquaglia, M. and Ferruci, J. 1984. Cytomegalovi rus- and Cryotosporidium-associated acalculous gangrenous cholecystitis. J. Med. 76: 1118-1123.
21. Boch, J., Gobel, E., Heine, J., Brandler U. and
Schloemer, L. 1982. Kryptosporidien-Infektion bei Haustie-
ren. Berl. Muench. Tieraerztl. Wochenschr, 95; 316-367.
22. Bogaerts, J., Lepage, P., Rouvroy, D. and Van-
depitte, J. 1984. Cryptosporidium spp., a frequent cause 157
of diarrhea in central Africa. J. Clin. Microbiol. 20: 874-
876.
23. Brady, E. M., Margolis, M. L. and Korzeniowski, 0.
M. 1984. Pulmonary cryptosporidiosis in acquired immune deficiency syndrome. J. Med. Assoc. 252: 89-90.
24. Bronsdon, M. A. 1984. Rapid dimethyl sulfoxide- modified acid-fast stain of Cryptosporidium oocysts in stool specimens. J. Clin. Microbiol. 19: 952-953.
25. Brownstein, D. G., Strandberg, J. D. and Montali,
R. J., Bush, M. and Fortner, J. 1977. Cryptosporidium in
snakes with hypertrophic gastritis. Vet. Pathol. 14; 606-
617.
26. Campbell, I., Tzipori, S., Hutchison, G. and
Angus, K. W. 1982. Effect of disinfectants on survival of
Cryptosporidium oocysts. Vet. Rec. Ill: 414-415.
27. Canning, E. U. and Anwar, M. 1968. Studies on
meiotic division in coccidial and malarial parasites. J.
Protozool. 15: 290-298.
28. Casemore, D. P., Armstrong, M. and Jackson, B.
1984. Screening for Cryptosporidium in stools. Lancet
1:734-735.
29. Casemore, D. P. and Jackson, B. 1983. Sporadic,
cryptosporidiosis in children. Lancet 11: 679. 158
30. Casemore, D. P. and Jackson, F. B. 1984. Hypoth esis: cryptosporidiosis in htaman beings is not primarily a zoonosis. J. Infection 9: 153-156.
31. Centers for Disease Control. 1982. Cryp tosporidiosis: assessment of chemotherapy of males with ac quired immune deficiency syndrome (AIDS). Morbid. Mortal.
Wkly. Repts. 31: 589-592.
32. Centers for Disease Control. 1984. Update:
Treatment of cryptosporidiosis in patients with acquired immune deficiency syndrome (AIDS). Morbid. Mortal. Wkly.
Repts. 33: 117-119.
33. Chobotar, B., and Scholtyseck, E. 1983. Ultra- structure. pp. 101-166. ^ Long, P. L., ed. The Biology of the Coccidia. University Park Press, Balitmore.
34. Clarke, J. J. 1895. A study of coccidia met with in mice. Q. J. Microsc. Sci. 37: 277-283.
35. Cockrell, B. Y., Valeric, M. G. and Garner, F. M.
1974. Cryptosporidiosis in the intestines of rhesus monkeys
(Macaca mulatta). Lab. Anim. Sci. 24: 881-887.
36. Collier, A. C., Miller, R. A. and Meyers, J. D.
1984. Cryptosporidiosis after marrow transplantation: per- son-to-person transmission and treatment with spiramycin.
Ann. Intern. Med. 101: 205-206. 159
37. Cross, R. F. and Moorhead, P. D. 1984. A rapid staining technic for Cryptosporidia. Mod. Vet. Pract. 65;
307.
38. Current, W. L. 1983. Human cryptosporidiosis.
N. Engl. J. Med. 309: 1326-1327.
39. Current, W. L. 1985. Cryptosporidiosis. J. Am.
Vet. Med. Assoc. 12: 1334-1338.
40. Current, W. L. and Haynes, T. B. 1984. Complete
development of Cryptosporidium in cell culture. Science
224: 603-605.
41. Current, W. L. and Long, P. L. 1983. Development
of human and calf Cryptosporidium in chicken embryos. J.
Infect. Pis. 148: 1108-1113.
42. Current, W. L. and Reese, N. C. 1986. A compari
son of endogenous development of three isolates of Cryp-
tosproidium in suckling mice. J. Protozool. 33: 98-108.
43a. Current, W. L., Reese, N. C., Ernst, J. V.,
Bailey, W. S., Heyman M. B. and Weinstein, W. M. 1983. Hu
man cryptosporidiosis in immunocompetent and immunodeficient
persons. N. Engl. J. Med. 308: 1252-1257.
43b. Current, W. L., Upton, S. J. and Haynes, T. B.
1986,. The life cycle of Cryptosporidium baileyi n. sp.
(Apicomplexa, Cryptosporidiidae) infecting chickens. J.
Protozool. 33: 289-296. 160
44. D'Antonio, R. G., Winn, R. E., Taylor, J. P., Gus- tafson, T. L., Current, W. L., Rhodes, M. M., Gary, G. W.
Jr., and Zajac, R. A. 1985. A waterborne outbreak of cryp- tosporidiosis in normal hosts- Ann. Intern. Med. 103: 886-
888.
45. Dhillon, A. S., Thacker, H. L., Dietzel, A. V. and
and Winterfield, R. W. 1981. Respiratory cryptosporidiosis
in broiler chickens. Avian Pis. 25: 747-751.
46. Doster, A. R,, Mahaffey, E. A. and McClearen, J.
R. 1979. Cryptosporidia in the cloacal coprodeum of Red-
lored parrots (Amazona autumnalis)• Avian Pis. 23: 654-661.
47. Eberhardt, U., Boch, J. and Heine, J. 1985.
Parasitologische und klinische Untersuchungen an experimen-
tell rait Kryptosporidien infizierten NMRI-Mausen. Berl.
Muench. Tieraerztl. Wochenschr. 98: 266-268.
48. Payer, R., Ernst, J. V., Miller, R. G. and Leek,
R. G. 1985. Factors contributing to clinical illness in
calves experimentally infected with a bovine isolate of
Cryptosporidium. Proc. Helminthol. Soc. Wash. 52: 64-70.
49. Payer, R., and Leek, R. G. 1984. The effects of
reducing conditions, medium, pH, temperature, and time on in
vitro excystation of Cryptosporidium. J. Protozool. 31:
567-569. 161
50. Fischer, 0. 1983. Attempted therapy and pro phylaxis of cryptosporidiosis in calves by administration of sulphadimidine. Acta Vet. BRNO 52: 183-190.
51. Fletcher, A., Sims, T. A. and Talbot, I. C. 1982.
Cryptosporidial enteritis without general or selective im mune deficiency. Br. Med. J. 285: 22-23.
52. Fletcher, 0. J., Munnell, J. F. and Page, R. K.
1975. Cryptosporidiosis of the bursa of Fabricius of chickens. Avian Pis. 19: 630-639.
53. Garcia, L. S., Bruckner, D. A., Brewer T. C. and
Shimizu, R. Y. 1983. Techniques for the recovery and iden tification of Cryptosporidium oocysts from stool specimens.
J. Clin. Microbiol. 18: 185-190.
54. Gardiner, C. H. and Imes, G. D. Jr. 1984. Cryp tosporidium sp. in the kidneys of a black-throated finch.
J. Vet. Med. Assoc. 185: 1401-1402.
55. Gibson, J. A., Hill, M. W. M. and Huber, M. J.
1983. Cryptosporidiosis in Arabian foals with severe com
bined immunodeficiency. Aust, Vet. J, 60: 378-379.
56. Glisson, J. R., Brown, T. P., Brugh, M., Page, R.
K., Kleven, S. H. and Davis, R. B. 1984. Sinusitis in
turkeys associated with respiratory cryptosporidiosis.
Avian Pis. 28: 783-790.
57. Goebel, E. and Braendler, U. 1982. Ultrastruc
ture of microgametogenesis, microgametes and gametogamy of 162
Cryptosporidium, sp. in the small intestine of mice. Protis- toloqica 18: 331-334.
58. Hampton, J. C. and Rosario, B. 1966. The attach ment of protozoan parasites to intestinal epithelial cells of the mouse. J. Parasitol. 52: 939-949.
59. Hart, C. A., Baxby, D. and Blundell, N. 1984.
Gastro-enteritis due to Cryptosporidium: a prospective sur vey in a children's hospital. J. Infection 9: 264-270.
60. Heine, J. 1982. Eine einfache Nachweismethode fur Kryptosporidien im Kot. Zentralbl. Veterinaermed. Reihe
B 29: 324-327.
61. Heine, J. and Boch, J. 1981. Kryptosporidien-
Infectionen beim Kalb. Nachweis, Vorkommen, und experimen- telle Ubertragung. Berl. Muench. Tieraerztl. Wochenschr.
94: 289-292.
62. Heine, J., Moon, H. W. and Woodmansee, D. B.
1984. Persistent Cryptosporidium infection in congenitally
athymic (nude) mice. Infect. Immun. 43: 856-859.
63. Heine, J., Moon, H. W., Woodmansee, D. B. and Poh-
lenz, J. F. L. 1985. Experimental tracheal and conjuncti
val infections with Cryptosporidium sp. in pigs. Vet.
Parasitol. 17: 17-25.
64. Heine, J., Pohlenz, J. F. L., Moon, H. W. and
Woode, G. N. 1984. Enteric lesions and diarrhea in 163
gnotobiotic calves monoinfected with Cryptosporidium spe cies. J. Infect. Pis. 150: 768-775^
65. Henriksen, Sv. Aa. and Pohlenz, J. F. L. 1981.
Staining of Cryptosporidia by a modified Ziehl-Neelsen tech nique. Acta Vet. Scand. 22: 594-596.
66. Hoerr, F. J., Ranck, F. M. Jr. and Hastings, T. F.
1978. Respiratory cryptosporidiosis in turkeys. J. Am.
Vet. Med. Assoc. 173: 1591-1593.
67. H0jlyng, N., M0lbak, K. and Jepsen, S. 1985.
Cryptosporidiosis in human beings is not primarily a zoonosis. J. Infection 11: 270-272.
68. H^jlyng, N., M^lbak, K., Jepsen, S. and Hansson,
A. P. 1984. Cryptosporidiosis in Liberian children. Lan
cet i: 734.
69. Holley, H. P. Jr. and Dover, C. 1986. Cryp
tosporidium: a common cause of parasitic diarrhea in other
wise healthy individuals. J. Infect. Dis. 153: 365-368.
70. Eolten-Andersen, W., Gerstoft, J., Henriksen, Sv.
Aa. and Pederson, N. S. 1984. Prevalence of Cryptosporidi
um among patients with acute enteric infections. J. Infec
tion 9: 277-282.
71. Hoover, D. M., Hoerr, F. J., Carlton, N. W., Kins
man, E. J. and Ferguson, H. W. 1981. Enteric cryp
tosporidiosis in a naso tang, Naso lituratus Bloch and
Schneider. J. Fish Dis. 4: 425-428. 164
72. Horen, W. P. 1983. Detection of Cryptosporidium in human fecal specimens. J. Parasitol. 69: 622-624.
73. Howerth, E. W. 1981. Bovine cryptosporidiosis.
J. S^. Afr. Vet. Assoc. 52: 251-253.
74. Inman, L. R. and Takeuchi, A. 1979. Spontaneous cryptosporidiosis in an adult female rabbit. Vet. Pathol.
16: 89-95.
75. Isaacs, D., Hunt,'G. H., Phillips, A. D., Price,
E. H., Raafat, F. and Walker-Smith, J. A. 1985. Cryp tosporidiosis in immunocompetent children. J. Clin. Pathol.
(Lond.) 38: 76-81.
76. Iseki, M. 1979. Cryptosporidium felis sp. n.
(Protozoa: Eimeriorina) from the domestic cat. Jpn. J.
Parasitol. 28: 285-307.
77. Itakura, C-, Goryo, M. and Umemura, T. 1984.
Cryptosporidial infection in chickens. Avian Pathol. 13:
487-499.
78. Itakura, C., Nakamura, H., Umemura, T. and Goryo,
M. 1985. Ultrastructure of cryptosporidial life cycle in chicken host cells. Avian Pathol. 14: 237-249.
79. Jensen, J. B. and Edgar, S. A. 1976. Possible secretory function of the rhoptries of Eimeria magna during penetration of cultured cells. J. Parasitol. 62: 988-992. 165
80. Jerrett, I. V., and Snodgrass, D. R. 1981. Cryp tosporidia associated with outbreaks of neonatal calf diar rhea. Aust. Vet. J. 57: 434-435.
81. Jervis, H. R., Merrill, T. G. and Sprinz, H.
1966. Coccidiosis in the guinea pig small intestine due to a Cryptosporidium. Am. J. Vet. Res. 27: 408-414.
82. Jokipii, A. M. M., Hemila, M. and Jokipii, L.
1985. Prospective study of acquisition of Cryptosporidium,
Giardia lamblia and gastrointestinal illness. Lancet ii:
487-489.
83. Jokipii, L., Pohjola, S. and Jokipii, A. M. M.
1983. Cryptosporidium; a frequent finding in patients with gastro-intestinal symptoms. Lancet ii: 358-360.
84. Jokipii, L., Pohjola, S. and Jokipii, A. M. M.
1985. Cryptosporidiosis associated with traveling and giar diasis. Gastroenterology 89; 838-842.
85. Kennedy, G- A., Kreitner, G. L. and Strafuss, A.
C. 1977. Cryptosporidiosis in three pigs. J. Vet.
Med. Assoc. 170; 348-350,
86. Kheysin, E. M. 1972. Life Cycles of Coccidia of
Domestic Animals. University Park Press, Baltimore.
87. Koch, K. L., Phillips, D. J., Aber, R. C. and Cur rent, W. L. 1985. Cryptosporidiosis in hospital personnel.
Ann. Intern. Med. 102; 593-596. 166
88. Koch, K. L., Shankey, T. V., Weinstein, G. S.,
Dye, R. E., Abt, A. B., Current, W. L. and Eyster, M. E.
1983. Cryptosporidiosis in a patient with hemophilia, com mon variable hypogammaglobulinemia, and the acquired im munodeficiency syndrome. Ann. Intern. Med. 99: 337-340.
89. Kocoshis, S. A., Cibull, M. L., Davis, T. E., Hin- ton, J. T., Seip, M. and Banwell, J. G. 1984. Intestinal and pulmonary cryptosporidiosis in an infant with severe combined immune deficiency. J. Pediatr. Gastroenterol.
Nutr. 3; 149-157.
90. Kovatch, R. M. and White, J. D. 1972. Cryp tosporidiosis in two juvenile rhesus monkeys. Vet. Pathol.
9: 426-440.
91. Lasser, K. H., Lewin, K. J. and Ryning, F. W.
1979. Cryptosporidia! enteritis in a patient with congeni tal hypogammaglobulinemia. Hum. Pathol. 10: 234-240.
92. Leek, R. G. and Payer, R. 1984. Prevalence of
Cryptosporidium infections, and their relation to diarrhea in calves on 12 dairy farms in Maryland. Proc. Helminthol.
Soc. Wash. 51: 360-361.
93. Lefkowitch, J. H., Krumholz, S., Feng-Chen, K.
-C., Griffin, P., Despommier D. and Brasitus, T. A. 1984.
Cryptosporidiosis in the human small intestine: a light and
electron microscopic study. Hum. Pathol. 15: 746-752. 167
94. Levine, N. D. 1971. Uniform terminology for the protozoan subphylum Apicomplexa. J. Protozool. 18: 352-355.
95. Levine, N. D. 1983. Taxonomy and life cycles of coccidia. pp. 1-33. ^ Long, P. L., ed. The Biology of the Coccidia. University Park Press, Baltimore.
96. Levine, N. D. 1984. Taxonomy and review of the coccidian genus Cryptosporidium (Protozoa, Apicomplexa). J.
Protozool. 31: 94-98.
97. Levine, N. D. 1984. The genera Cryptosporidium and Epieimeria in the coccidian family Cryptosporidiidae.
Trans. Am. Microsc. Soc. 103: 205-206.
98. Levine, N. D., Corliss, J. 0., Cox, F. E. G.,
Deroux, G., Grain, J., Honigberg, B. M., Leedale, G. F.,
Loeblich, A. R. Ill, Lom, J., Lynn, D., Merinfeld, Z. G.,
Page, F. C., Poljansky, G., Sprague, V., Varva, J. and
Wallace, F. G. 1980. A newly revised classification of the protozoa. J. Protozool. 27: 37-58.
99. Lewis I. J., Hart, C. A. and Baxby, D. 1985.
Diarrhoea due to Cryptosporidium in acute lymphoblastic leukaemia. Arch. Pis. Child. 60: 60-62.
100. Lindsay, D. S., Blagburn, B. L., Sundermann, C.
A., Hoerr F. J. and Ernest, J. A. 1986. Experimental Cryp tosporidium infections in chickens; oocyst structure and tissue specificity. J. Vet. Res. 47: 876-879. 168
101. Links, I. J. 1982. Cryptosporidial infection of piglets. Aust. Vet. J. 58: 60-62.
102. Ma, P., Kaufman, D. L., Hemlick, C. G., D'Souza,
A. J. and Navin, T. R. 1985. Cryptosporidiosis in tourists returning from the Caribbean. N. Engl. J. Med. 312: 647-
648.
103. Ma, P. and Soave, R. 1983. Three-step stool examination for cryptosporidiosis in 10 homosexual men with protracted watery diarrhea. J. Infect. Pis. 147; 824-828.
104. Mason, R. W. and Hartley, W. J. 1980. Respira tory cryptosporidiosis in a peacock chick. Avian Pis. 24;
771-776.
105. Marcial, M. A. and Madara, J. L. 1986. Cryp tosporidium: cellular localization, structural analysis of absorptive cell-parasite membrane-membrane interactions in guinea pigs, and suggestion of protozoan transport by M cells. Gastroenterology 90; 583-594.
106. Mata, L., Bolanos, H., Pizarro, P. and Vives, M.
1984. Cryptosporidiosis in children from some highland Cos ta Rican rural and urban areas. J. Trop. Med. Hyg. 33:
24-29.
107. Matovelo, J. A., Landsverk, T. and Amaya-Posada,
G. 1984. Cryptosporidiosis in Tanzanian goat kids; scan
ning and transmission electron microscopic investigations.
Acta Vet. Scand. 25: 322-326. 169
108. McNabb, S. J. N.; Hensel, D. M., Welch, D. F.,
Heijbel, H., McKee, G- L., and Istre, G. R. 1985. Compari son of sedimentation and flotation techniques for iden tification of Cryptosporidium sp. oocysts in a large out break of human diarrhea. J. Clin. Microbiol. 22 ;587-589.
109. Meisel, J. L., Perera, D. R., Meligro, C. and
Rubin, C. E. 1976. Overwhelming watery diarrhea associated with a Cryptosporidium in an immunosupressed patient. Gas troenterology 70: 1156-1160.
110. Meuten, D. J., Van Kruiningen, H. J. and Lein, D.
H. 1974. Cryptosporidiosis in a calf. J. Vet. Med.
Assoc. 165: 914-917.
111. Miller, R. A., Holmberg, R. E. Jr. and Clausen,
C. R. 1983. Life-threatening diarrhea caused by Cryp tosporidium in a child undergoing therapy for acute lym phocytic leukemia. J. Pediatr. 103: 256-259.
112. Montessori, G. A. and Bischoff, L. 1985. Cryp
tosporidiosis: a cause of summer diarrhea in children. Can.
Med. Assoc. J. 132: 1285.
113. Moon, H. W. and Bemrick, W. J. 1981. Fecal
transmission of calf Cryptosporidia between calves and pigs.
Vet. Pathol. 18: 248-255.
114. Moon, H. W., McClurkin, A. W., Isaacson, R. E.,
Pohlenz, J,, Skartvedt, S. M., Gillette, K. G. and Baetz, A. 170
L. 1978. Pathogenic relationships of rotavirus, Escheri chia coli, and other agents in mixed infections in calves.
J. Vet. Med. Assoc. 173; 577-583.
115. Moon, H. W., Schwartz, A., Welch, M. J., McCann,
P. P. and Runnels, P. L. 1982. Experimental fecal trans mission of human Cryptosporidia to pigs, and attempted treatment with an ornithine decarboxylase inhibitor. Vet.
Pathol. 19: 700-707.
116. Moon, H. W., Woode, G. N. and Ahrens, F. A.
1982. Attempted chemoprophylaxis of cryptosporidiosis in calves. Vet. Rec. 110 ;181.
117. Morin, M., Lariviere, S. and Lallier, R. 1976.
Pathological and microbiological observations made on spon taneous cases of acute neonatal calf diarrhea. Can. J.
Comp. Med. 40: 228-240.
118. Navin, T. R. and Juranek, D. D. 1984. Cryp
tosporidiosis: clinical, epidemiologic,- and parasitologic
review. Rev. Infect. Pis. 6; 313-327.
119. Nichols, G. and Thorn, B. T. 1984. Screening for
Cryptosporidium in stools. Lancet i: 735.
120. Nime, F. A., Burek, J. D., Page, D. L., Holscher,
M. A. and Yardley, J. H. 1976. Acute.enterocolitis in a
human being infected with the protozoan Cryptosporidium.
Gastroenterology 70: 592-598. 171
121. Panciera, R. J., Thomassen, R. W., and Garner, F.
M. 1971. Cryptosporidial infection in a calf. Vet.
Pathol. 8; 479-484.
122. Pavlasek, I. 1981. First record of Cryp tosporidium sp. in calves in Czechoslovakia. Folia Parasi- tol. (Prague) 28: 187-189.
123. Pavlasek, I. 1982. First detection of Cryp tosporidium sp. oocysts in calf faeces by floatation method.
Folia Parasitol. (Prague) 29: 115-118.
124. Pavlasek, I. 1982. The occurrence of Cryp tosporidium sp. in emergency-slaughtered calves and the course of excretion of the protozoan oocysts in calves on two farms in the South Bohemian Region. Vet. Med. (Prague)
27: 729-740.
125. Pavlasek, I. 1983. Experimental infection of
cat and chicken with Cryptosporidium sp. oocysts isolated
from a calf. Folia Parasitol. (Prague) 30: 121-122.
126. Pavlasek, I- and Mares, J. 1983. The effect of
single disinfection of a farm on the course of cryp-
tosporidiosis infections in calves. Vet. Med. (Prague) 28:
449-455.
127. Pavlasek, I. and Nikitin, V. F. 1983. Finding
of Cryptosporidium sp. in calves in the USSR. Folia Parasi
tol. (Prague) 30: 4. 172
128. Payne, P., Lancaster, L. A., Heinzman, M. and
McCutchan, J. A. 1983. Identification of Cryptosporidium in patients with the acquired immunodeficiency syndrome. N.
Engl. J. Med. 309: 613-614.
129. Pearson, G. R. and Logan, E. F. 1978. Demon stration of Cryptosporidia in the small intestine of a calf by light, transmission electron and scanning electron microscopy. Vet. Rec. 103: 212-213.
130. Pearson, G. R. and Logan, E. F. 1983. Scanning and transmission electron microscopic observations on the host-parasite relationship in intestinal cryptosporidiosis of neonatal calves. Res. Vet. Sci. 34: 149-154.
131. Pearson, G. R., Logan, E. F. and McNulty, M. S.
1982. Distribution of Cryptosporidia within the gastroin testinal tract of young calves. Res. Vet. Sci. 33: 228-231.
132. Peeters, J. E., Van Openbosch, E. and Glorieux,
B. 1982. Demonstration of Cryptosporidia in calf faeces: a comparative study. Vlaams Diergeneeskd. Tijdschr. 51: 513-
523.
133. Perez-Schael, I., Boher, Y., Mata, L., Perez, M. and Tapia, F. J. 1985. Cryptosporidiosis in Venezuelan children with acute diarrhea. J. Trop. Med. Hyg. 34:
721-722. 173
134. Pitlik, S. D., Fainstein, V., Rios, A., Guarda,
L., Mansell, P. W. A. and Hersh, E, M. 1983. Cryp tosporidia! cholecystitis. N. Engl. J. Med. 308: 967.
135. Pohlenz, J., Bemrick, W. J., Moon, H. W. and Che ville, N. F. 1978. Bovine cryptosporidiosis: a transmis sion and scanning electron microscopic study of some stages in the life cycle and of the host-parasite relationship.
Vet. Pathol. 15: 417-427.
136. Pohlenz, J., Moon, H. W., Cheville N. F. and Bem rick, W. J. 1978. Cryptosporidiosis as a probable factor in neonatal diarrhea of calves. J. Vet. Med. Assoc.
172: 452-457.
137. Portnoy, D., Whiteside, M. E., Buckley E. Ill and
McLeod, C. L. 1984. Treatment of intestinal cryp tosporidiosis with spiramycin. Ann. Intern. Med. 101: 202-
204.
138. Powell, H. S., Holscher, M. A., Heath J. E. and
Beasley, F. F. 1976. Bovine cryptosporidiosis (a case re
port). Vet. Med. Small Amin. Clin. 71: 205-207.
139. Proctor, S. J. and Kemp, R. L. 1974. Cryp
tosporidium ansinum sp. n. (Sporozoa) in a domestic goose
Anser anser L. from Iowa. J. Protozool. 21: 664-666.
140. Rahaman, A. S. M. H., Sanyal, S. C., Al-Mahmud,
K. A., Sobhan, A., Hossain K. S. and Anderson, B. C. 1984. 174
Cryptosporidiosis in calves and their handlers in
Bangladesh. Lancet ii: 221.
141. Randall, C. J. 1982. Cryptosporidiosis of the bursa of Fabricius and trachea in broilers. Avian Pathol.
11: 95-102.
142. Randall, C. J. 1986. Conjunctivitis in pheas ants associated with cryptosporidial infection. Vet. Rec.
118: 211-212.
143a. Ratnam, S., Paddock, J., McDonald, E., Whitty,
D., Jong, M. and Cooper, R. 1985. Occurrence of Cryp tosporidium oocysts in fecal samples submitted for routine microbiological examination. J. Clin. Microbiol. 22: 402-
404.
143b. Reduker, D. W. and Speer, C. A. 1985. Factors
influencing excystation in Cryptosporidium oocysts from cat
tle. J. Parasitol. 71: 112-115.
144. Reduker, D. W., Speer, C. A. and Blixt, J. A.
1985. Ulrastructural changes in the oocyst wall during ex
cystation of Cryptosporidium parvum (Apicomplexa; Eucoc-
cidiorida). Can. J. Zool. 63: 1892-1896.
145. Reduker, D. W., Speer, C. A. and Blixt, J. A.
1985. Ultrastructure of Cryptosporidium parvum oocysts and
excysting sporozoites as revealed by scanning electron
microscopy. J. Protozool. 32: 708-711. 175
146. Reese, N. C., Current, W. L., Ernst, J. V. and
Bailey, W. S. 1982. Cryptosporidiosis of man and calf: a case report and results of experimental infections in mice and rats. J. Trop. Med. Hyg. 31: 226-229.
147. Rehg, J. E., Lawton, G. W. and Pakes, S. P.
1979. Cryptosporidium cuniculus in the rabbit (Oryctolaqus cuniculus). Lab. Anim. Sci. 29: 656-660.
148. Ribeiro, C. D. and Palmer, S. R. 1986. Family outbreak of cryptosporidiosis. Br. Med. J. 292: 337.
149. Ryley, J. F. 1973. Cytochemistry, physiology and biochemistry, pp. 145-182. ^ Hammond, D. M. and Long,
P. L., eds. The Coccidia. University Park Press,
Baltimore.
150. Ryley, J. F., Bentley, M., Manners, D. J. and
Stark, J. R. 1969. Amylopectin, the storage polysaccharide of the coccidia Eimeria burnetti and E. tenella. J. Parasi- tol. 55: 839-845.
151. Sanford, S. E. and Josephson, G. K. A. 1982.
Bovine cryptosporidiosis: clinical and pathological findings
in forty-two scouring neonatal calves. Can. Vet. J. 23:
343-347.
152. Schmidt, U. and Nienhoff, H. 1982. Kryp-
tosporidiose beim Schwein. Dtsch. Tieraerztl. Wochenschr.
89: 437-439. 176
153. Schmitz, J. A. and Smith, D. H. 1975. Cryp tosporidium infection in a calf. J. Vet. Med. Assoc.
167: 731-732.
154. Scholtyseck, E., Rommel, A. and Heller, G. 1969.
Licht- und elektronenmikroskopishe Untersuchungen zur Bil- dund der Oocystenhulle bei Eimerien (Eimeria perforans, E. stiedae und E. tenella). Z, Parasitenkd. 31; 289-298.
155. Schultz, M. G. 1983. Emerging zoonoses. N.
Engl. J. Med. 308: 1285-1286.
156. Shahid, N. S., Rahman, A. S. M, H., Anderson, B.
C., Mata, L. J. and Sanyal, S. C. 1985. Cryptosporidiosis
in Bangladesh. Br. Med. J. 290: 114-115.
157. Sherwood, D., Angus, K. W., Snodgrass D. R. and
Tzipori, S- 1982. Experimental cryptosporidiosis in
laboratory mice. Infect. Immun. 38: 471-475.
158- Skeels, M. R., Sokolow, R., Hubbard, C. V. and
Foster, L. R.. 1986. Screening for coinfection with Cryp
tosporidium and Giardia in Oregon public health clinic pa
tients. J. Public Health 76: 270-273.
159. Slavin, D. 1955. Cryptosporidium meleagridis
(sp. nov.). J. Comp. Pathol. 65: 262-266.
160. Sloper, K. S., Dourmashkin, R. R., Bird, R. B.,
Slavin, G. and Webster, A. D. B. 1982. Chronic malabsorp
tion due to cryptosporidiosis in a child with immunoglobulin
deficiency. Gut 23: 80-82. 177
161. Snodgrass, D. R., Angus, K. W. and Gray, E. W.
1984. Experimental cryptosporidiosis in germfree lambs. J.
Comp. Pathol. 94; 141-152.
162. Snodgrass, D. R., Angus, K. W., Grpy, E. W.,
Keir, W. A. and Clerihew, L. W. 1980. Cryptosporidia as sociated with rotavirus and an Escherichia coli in an out break of calf scour. Vet. Rec. 106: 458-460.
163. Snyder, S. P., England, J. J. and McChesney, A.
E. 1978. Cryptosporidiosis in immunodeficient Arabian foals. Vet. Pathol. 15: 12-17.
164. Soave, R. and Ma, P. 1985. Cryptosporidiosis.
Traveler's diarrhea in two families. Arch. Intern. Med.
145: 70-72.
165. Stemmermann, G. N., Hayashi, T., Glober, G. A.,
Oishi N. and Frankel, R. 1980. Cryptosporidiosis. Report of a fatal case complicated by disseminated toxoplasmosis.
J. Med. 69: 637-642.
166. Sterling, C. R., Seegar, K. and Sinclair, N. A.
1986. Cryptosporidium as a causative agent of traveler's
diarrhea. J. Infect. Pis. 153: 380-381.
167. Szabo, J. R. and More, K. 1984. Cryp
tosporidiosis in a snake. Vet. Med. Small Anim. Clin., 79:
96-98. 178
168. Tarwid, J. N., Cawthorn, R. J. and Riddell, C.
1985. Cryptosporidiosis in the respiratory tract of turkeys in Saskatchewan. Avian Pis. 29: 528-532.
169. Taylor, J. P., Perdue, J, N., Dingley, D., Gus- tafson, T. L., Patterson, M. and Reed, L. A. 1985. Cryp tosporidiosis outbreak in a day-care center. J. Pis.
Child. 139: 1023-1025.
170. Tham, V. L., Kniesberg, S. and Pixon, B. R.
1982. Cryptosporidiosis in quails. Avian Pathol. 11; 619-
626.
171. Triffit, M. J. 1925. Observations on two new
species of Coccidia parasitic in snakes. Protozoology 1:
19-26.
172. Tyzzer, E. E. 1907. A sporozoan found in the
peptic glands of the common mouse- Proc. Soc. Exp. Biol.
Med. 5: 12-13.
173. Tyzzer, E. E. 1910. An extracellular coccidium,
Cryptosporidium muris (gen. et sp. nov.) of the gastric
glands of the common mouse. J. Med. Res. 23: 487-509.
174. Tyzzer, E. E. 1912. Cryptosporidium parvum (sp.
nov.) a coccidium found in the small intestine of the common
mouse. Arch. Protistenkd. 26: 394-412.
175. Tzipori, S. 1983. Cryptosporidiosis in animals
and humans. Microbiol. Rev. 47: 84-96. 179
176. Tzipori, S., Angus, K. W., Campbell I. and
Clerihew, L. W. 1981. Diarrhea due to Cryptosporidium in fection in artificially reared lambs. J. Clin. Microbiol.
14: 100-105.
177. Tzipori, S., Angus, K. W., Campbell, I. and Gray,
E. W. 1980. Cryptosporidium: evidence for a single-species
genus. Infect. Immun. 30: 884-886.
178. Tzipori, S., Angus, K. W., Campbell, I. and Gray,
E. W. 1982. Experimental infection of lambs with Cryp
tosporidium isolated from a patient with diarrhoea. Gut 23:
71-74.
179. Tzipori, S., Angus, K. W., Campbell, I. and Sher
wood, D. 1981. Diarrhea in young red deer associated with
infection with Cryptosporidium. J. Infect. Dis. 144: 170-
175.
180. Tzipori, S., Angus, K. W., Gray, E. W. and Camp
bell, I. 1980. Vomiting and diarrhea asociated with cryp-
tosporidial infection. N. Engl. J. Med. 303: 818.
181. Tzipori, S., Campbell, I., Sherwood, D., Snod-
grass, D. R. and Whitelaw, A. 1980. An outbreak of calf
diarrhoea attributed to Cryptosporidia! infection. Vet.
Rec. 107: 579-580.
182. Tzipori, S., Larsen, J., Smith, M. and Luefl, R.
1982. Diarrhoea in goat kids attributed to Cryptosporidium
infection. Vet. Rec. Ill: 35-36. 180
183. Tzipori, S., McCartney, E., Lawson, G. H. K.,
Rowland. A. C. and Campbell, I. 1981. Experimental infec tion of piglets with Cryptosporidium• Res. Vet. Sci. 31:
358-369.
184. Tzipori, S., Sherwood, D., Angus, K. W., Camp bell, I. and Gordon, M. 1981. Diarrhea in lambs: ex perimental infections with enterotoxigenic Escherichia coli, rotavirus, and Cryptosporidium sp. Infect. Immun. 33: 401-
406.
185. Tzipori, S., Smith, M., Birch, C., Barnes, G. and
Bishop, R. 1983. Cryptosporidiosis in hospital patients with gastroenteritis, to. J. Trop. Med. Hyg. 32: 931-934.
186. Tzipori, S., Smith, M., Kalpin, C., Angus, K. W.,
Sherwood, D. and Campbell, I. 1983. Experimental cryp tosporidiosis in calves: clinical manifestations and pathological findings. Vet. Rec. 112: 116-120.
187. Tzipori, S., Smith, M., Makin, T. and Halpin, C.
1982. Enterocolitis in piglets caused by Cryptosporidium sp. purified from calf faeces. Vet. Parasitol. 11: 121-126.
188. Upton, S. J. and Current, W. L. 1985. The spe cies of Cryptosporidium (Apicomplexa: Cryptosporidiidae) infecting mammals. J. Parasitol. 71: 625-629.
189. Vetterling, J. M., Jervis, H. R., Merrill, T. G.
and Sprinz, H. 1971. Cryptosporidium wrairi sp. n. from 181
the guinea pig Cavia porcellus, with an emendation of the genus. J, Protozool. 18: 243-247.
190. Vetterling, J. M., Takeuchi, A- and Madden, P. A.
1971. Ultrastructure of Cryptosporidium wrairi from the guinea pig. J. Protozool. 18: 248-260.
191. Wang, C. C. 1983. Biochemistry and physiology of coccidia. pp. 167-228. ^ Long, P. L., ed. The Biol ogy of the Coccidia. University Park Press, Baltimore.
192. Weikel, C. S., Johnston, L. I., De Sousa, M. A. and Guerrant, R. L. 1985. Cryptosporidiosis in northeast ern Brazil; association with sporadic diarrhea. J. Infect.
Dis. 151: 963-965.
193. Weisburger, W. R., Hutcheon, D. F., Yardley, J.
H., Roche, J. C., Hillis, W. D. and Charache, P. 1979.
Cryptosporidiosis in an imiaunosuppressed renal-transplant recipient with IgA deficiency. J. Clin. Pathol. 72:
473-478.
194. Wenyon, C. M. 1907. Observations on the proto zoa in the intestine of mice. Arch = Protistenkd. (Supp. i);
169.
195. Whittington, R. J. and Wilson, J. M. 1985.
Cryptosporidiosis of the respiratory tract in a pheasant.
Aust. Vet. J. 62: 284.
196. Willson, P. J. and Acres, S. D. 1982. A com
parison of dichromate solution flotation and fecal smears 182
for diagnosis of cryptosporidiosis in calves. Can. Vet. J.
23: 240-246.
197. Wilson, D. W., Day, P. A. and Brummer, M. E. G.
1984. Diarrhea associated with Cryptosporidium spp. in juvenile macaques. Vet. Pathol. 21: 447-450.
198. Wolfson, J. S., Hopkins, C. C., Weber, D. J.,
Richter, J. M., Waldron, M. A. and McCarthy, D. M. 1984.
An association between Cryptosporidium and Giardia in stool.
N. Engl. J. Med. 310: 788.
199. Wolfson, J. S., Richter, J. M., Waldron, M. A.,
Weber, D. J., McCarthy D. M. and Hopkins, C. C. 1985.
Cryptosporidiosis in immunocompetent patients. N. Engl. J.
Med. 312: 1278-1282.
200. Woodmansee, D. B. and Pohlenz, J. F. L. 1983.
Development of Cryptosporidium sp. in a human rectal tumor
cell line. pp. 306-317. ^ Proceedings of the Fourth
International Symposium on Neonatal Diarrhea. Veterinary
Infectious Disease Organization, Saskatoon.
201= Wright, P= A., Harrison, J. M. and Byrom, I.
1984. Cryptosporidiosis. Br. Med. J. 289: 1148.
202. Zar, F., Geiseler, P. J. and Brown, V. A. 1985.
Asymptomatic carriage of Cryptosporidium in the stool of a
patient with the acquired immunodeficiency syndrome. J.
Infect. Dis. 151: 195. 183
203. Zierdt, W. S. 1984. Concentration and iden tification of Cryptosporidium sp. by use of a parasite con centrator. J. Clin. Microbiol. 20: 860-861. 184
ACKN0WLEDGÎ4ENTS
I am most grateful to the large number of people who have helped me in the work described in this dissertation.
In particular, I wish to acknowledge the cooperative efforts of Drs. Edwin Powell, Joachim Pohlenz, and Harley Moon.
They have done more for me than I can remember, and I would have been unable to do any of this work without their sup port and assistance. Thanks to Drs. Sheldon Shen and Warren
Dolphin who were always helpful, supportive and friendly as members of the graduate committee.
Special thanks to the whole crew in B-14; Mayo, Gary,
Paul, Tom, Jim, Bruce, Rob, Doug, and Becky have all helped me immensely at one time or another. Deb Hainmen should be singled out for the great job she did for me in the ell-too- brief time we worked together. Thanks to Jane, Sally, and
Joan in the Vet Path EM lab ; Wayne, Gene, and Tom in the
NADC Photo Lab; Kathleen in the front office; Karen and Phil in B-8; Gary, Don, Roger, and John in Animal Care; Pete in
Animal Supply; Keeta and Ron in Office Services; and to everyone else who lent a helping hand.
Financial support was provided by a cooperative agree
ment between the United Stated Department of Agriculture and
Iowa State University and by a fellowship from the Salsbury
Foundation. I am indebted to Dr. Philip 0'Berry and Dr. 185
William Switzer for providing for the administration of these funds.
Finally, I would like to acknowledge the loving support of my wife, Valerie, and my parents, who showed infinite patience with my seemingly endless education. This disser tation is dedicated to my grandmother, Martha Woodmansee, who I miss very much.