The Genus Cystoisospora was created in 1977 (1) to account for the recently described
facultative two-host life cycles observed occurring in feline and canine Isospora
species (2, 3). The justification for the creation of the Genus Cystoisospora and
placement of mammalian Isospora species in it was based on morphological, biological,
and genetic differences between Isospora species from non-mammalian hosts and mammalian
hosts (4–6). The orally infectious tissue cyst stage present in the intermediate host
contains a single asexual stage and is referred to as a monozoic tissue cyst (MZTC)
because it contains a single organism within a parasitophorous vacuole surrounded
by a tissue cyst wall (7–9). The stage is classified as a zoite because it has all
the ultrastructural features found in sporozoites, tachyzoites, and bradyzoites of
other Sarcocystidae. Most notably the zoite contains a crystalloid body observed in
sporozoites of Cystoisospora species (10). Crystalloid bodies are spherical accumulations
of granular cytoplasmic inclusions similar to beta-glycogen particles. They are in
the same location of the sporozoite as are refractile bodies of Eimeria species sporozoites
excysted from oocysts (11) and occasionally early generation merozoites of Eimeria
species (12). Crystalloid bodies and refractile bodies are believed to serve as sites
of energy storage to maintain the dormant sporozoites until they are ingested by a
host and become metabolically active. The presence of crystalloid bodies in sporozoites
in oocysts and zoites in MZTC of Cystoisospora is now recognized as a feature useful
in their identification (6).
It is not known if zoites originate from oocysts that have excysted and represent
sporozoites that migrate to extra-intestinal sites without developing or if they represent
a post divisional stage, such as a tachyzoite, bradyzoite, or merozoite. This remains
one of the most important questions concerning the biology of this group and has important
implications in the management of C. belli infections in immune compromised patients.
However, the tremendous number of MZTC each containing a single zoite observed in
tissues of immune suppressed patients with C. belli infections indicates that zoites
in MZTC represent an asexual stage that has been produced by multiplication either
by endodyogeny or another form of merogony and has migrated to its location of encystation
and became a MZTC (13).
Pioneering studies on the development of Cystoisospora species in cell cultures were
conducted by Dr. Ron Fayer at the United States Department of Agriculture in Beltsville,
Maryland, from 1972 to 1974 (14–16). These studies demonstrated that sporozoites excysted
from oocysts of C. rivolta and C. felis collected from cats, and sporozoites of C.
canis from dogs, entered cultured cells and divided repeatedly by endodyogeny. They
provided a foundation for future studies of the medically important C. belli from
humans and economically important C. suis in young swine.
Cystoisospora rivolta developed in feline kidney cells, embryonic bovine kidney cells,
and Madin-Darby canine kidney (MDCK) cells (14). Development was delayed by 24 h and
was minimal in in MDCK cells. Cystoisospora felis developed in embryonic intestine,
esophageal epithelium, amnion, lung, and Hela cells from humans, kidney cells from
chickens, embryonic tracheal cells from bovines, and MDCK cells from dogs but cells
from cats were not examined (16). Development by C. felis was similar in all these
cell types.
Development of sporozoites of C. canis was examined in MDCK cells and primary cells
from embryonic canine kidneys, and embryonic canine intestine as well as embryonic
bovine trachea and kidney (15). Development by endodyogeny occurred in all host cell
types 3–4 days after inoculation. A study of development of C. canis sporozoites using
bovine turbinate and African green monkey kidney (CV-1) cells provided remarkedly
different findings of no asexual multiplication but rather MZTC formation in both
host cell types (17). Examination of these stages using transmission electron microscopy
confirmed they were MZTC and that they were similar to C. belli MZTC in the tissues
of humans (13, 18), C. canis in mice (9), C. ohioensis in mice (8) and C. felis in
mice (19). The organisms in these MZTC all contain crystalloid bodies when viewed
using transmission electron microscopy. An in-depth study was conducted on the biology
of MZTC of C. canis using two canine, four human, one monkey, and one bovine cell
lines (20). No asexual multiplication occurred in any cell line however MZTC were
produced in all cell lines. It is important to note that MDCK cells were used by both
Fayer and Mahrt in their study (15) and Houk and Lindsay in their study (20) with
dramatically different results. When host cells containing MZTC were exposed to excystation
solution (0.75% sodium taurocholic acid and 0.25% trypsin solution) the sporozoites
became motile and exited the MZTC and were able to infect new host cells and produce
MZTC again. The same events occurred, except the tissue cyst wall was dissolved by
the acid-pepsin solution, when MZTC were exposed to acid-pepsin solution suggesting
survival through the stomach and oral infectiousness of this stage (20). Additional
research is needed to account for these different findings between these three C.
canis isolates by different research groups. It suggests that two different genetic
types of C. canis exist and that one divides by endodyogeny in culture and the other
produces MZTC. How this would translate to in vivo development and pathogenicity in
dogs is also an interesting area needing research.
The initial studies on the development of C. suis in five types of mammalian host
cells in culture indicated that development was by endodyogeny (21) and stages were
structurally similar to Type I meronts observed in neonatal pigs (22). Similar findings
were observed in primary porcine kidney cells and primary embryonic bovine kidney
cells (23). However, stages suggestive of multinucleate Type II meronts were seen
in primary porcine kidney cells but no merozoites were produced (23). The ultrastructural
aspects of C. suis development of Type I meronts by endodyogeny in these cell cultures
(24) was examined and the findings were not different from those reported to occur
in the small intestinal epithelial cells of infected pigs (25). Notably crystalloid
bodies were seen in Type I merozoites in vitro (24). Complete development of C. suis
was observed in a swine testicular cell line (26). Development was delayed 2–4 days
and most stages were Type I meronts and merozoites. The ultrastructure of microgamonts,
microgametes, and oocysts were described (26). Oocysts did not sporulate in culture.
Studies conducted in 2013 using a porcine intestinal epithelial cell line reported
similar results (27). The study demonstrated that high densities of gamonts occurred
at 12 days after infection and found that an optimum infective dose was one sporozoite
per 100 host cells (27).
No evidence of MZTC formation has been reported in cell cultures. Similarly, studies
in pigs and potential paratenic hosts have been negative (28, 29). This is puzzling
because the biology of C. suis conforms to all other reported features of the genus.
Four studies have examined development of sporozoites obtained from oocysts of C.
belli in human and other mammalian cell cultures (30–33). The studies have demonstrated
that development occurs in several cell types from humans including those derived
from human ileocecal adenocarcinoma (HCT-8), epithelial carcinoma of the lung, and
macrophages. Other mammalian cells examined have consisted of mouse macrophages, Madin-Darby
bovine kidney, Rhesus monkey kidney (RMK) and African green monkey kidney (VERO) cells.
Endodyogeny was described in HCT-8 cells and RMK cells using transmission electron
microscopy (32). The authors found no crystalloid bodies in merozoites of C. belli
as are present in C. suis (24) while undergoing endodyogeny in vitro but otherwise
the structural findings were similar to those reported for C. suis. Stages of C. belli
obtained from macrophage cell cultures were infectious for RMK cells upon subinoculation
and development by endodyogeny occurred (31) but MZTC were not reported.
I have attempted to point out the similarities and important differences in the development
of Cystoisospora species from mammals and to provide insight into future research
directions. Development will occur in many host cell types but studies with C. suis
illustrate that selection of the proper host cell type can lead to complete development
thus increasing the utility of an in vitro system. Studies with C. canis suggest that
genetic differences in Cystoisospora species exist that are reflected by the phenotype
of development expressed in vitro. I look forward to seeing what advances in our knowledge
of this group are revealed in the future.
Author Contributions
The author confirms being the sole contributor of this work and has approved it for
publication.
Conflict of Interest Statement
The author declares that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.