Endogenous development of Cystoisospora belli in intestinal and biliary epithelium of humans.

Cystoisospora (Isospora) belli is a coccidian parasite of humans. It can cause serious digestive disorders involving infection of intestines, biliary tract and gallbladder, especially in those with depressed immunity. It has a direct fecal-oral transmission cycle. After ingestion of sporulated oocysts, the parasite multiplies asexually and sexually within host epithelial cells, resulting in unsporulated oocysts that are excreted in feces. The details of asexual and sexual stages are not known and certain inclusions in epithelial cells in biopsy samples have been erroneously identified recently as C. belli. Here, we provide details of developmental stages of C. belli in two patients, in duodenal biopsy of one and biliary epithelium of the other. Immature and mature asexual stages (schizonts/meronts) were seen in epithelial cells. The merozoites were seen singly, in pairs and in groups in single parasitophorous vacuole (pv) in host cytoplasm. Immature and mature meronts were seen together in the same pv; up to eight nuclei were seen in meronts that retained elongated crescent shape; round multinucleated schizonts, seen in other coccidians, were not found. Meronts were up to 25 µm long and contained up to ten merozoites that were 8-11 µm long. The merozoites and meronts contained PAS-positive granules. Microgamonts (male) contained up to 30 nuclei that were arranged at the periphery and had condensed chromatin; 1-3 PAS-positive, eosinophilic, residual bodies were left when microgametes were formed. The microgametes were 4 µm long and PAS-negative. All stages of macrogamonts, including oocysts were PAS-positive. The detailed description of the life cycle stages of C. belli reported here should facilitate in histopathologic diagnosis of this parasite.

Isospora suis in an epithelial cell culture system - an in vitro model for sexual development in coccidia.

Coccidian parasites are of major importance in animal production, public health and food safety. The most frequently used representative in basic research on this group is Toxoplasma gondii. Although this parasite is well investigated there is no adequate in vitro model for its sexual development available and knowledge on this important life cycle phase is therefore scarce. The use of Isosporasuis, a sister taxon to T. gondii and the causative agent of piglet coccidiosis, could provide a solution for this. In the present study an in vitro model for neonatal porcine coccidiosis in cells representative for the in vivo situation in the piglet gut was developed and evaluated. The parasite development was investigated by light and transmission electron microscopy and optimum culture conditions were evaluated. Intestinal porcine epithelial cells (IPEC-J2) adequately representing the natural host cells supported the development of all endogenous life cycle stages of I. suis, including gametocytes and oocysts. A concentration of 5% fetal calf serum in the culture medium led to highest gametocyte densities on day 12 post infection. Low infection doses (≤1 sporozoite for 100 host cells) were best for oocyst and gametocyte development. The presented system can also be used for immunostaining with established antibodies developed against T. gondii (in our case, anti-TgIMC3 antibodies directed against the inner membrane complex 3). The complete life cycle of I. suis in a cell line representing the natural host cell type and species provides a unique model among coccidian parasites and can be used to address a wide range of topics, especially with regard to the sexual development of coccidia.

Autofluorescence microscopy for the detection of nematode eggs and protozoa, in particular Isospora suis, in swine faeces.

Parasites from swine faeces were examined for autofluorescence. Oocysts of Eimeria polita, E. scabra and Isospora suis, cysts of Balantidium coli and eggs of Oesophagostomum dentatum, Strongyloides ransomi and Trichuris suis (but not those of Ascaris suum) emitted light after excitation with UV light. I. suis oocyst counts in McMaster chambers utilising autofluorescence were compared to those from conventional bright field microscopy. Similarly, faecal smears containing I. suis were examined using the same techniques. Autofluorescence was superior to bright field microscopy in detecting oocysts after flotation and was highly significantly more sensitive when direct smears were examined.

Re-descriptions of Isospora ameivae Carini, 1932 in the Teiid Lizard Ameiva ameiva and Isospora hemidactyli Carini, 1936 in the Gecko Hemidactylus mabouia, with Particular Reference to their Endogenous Stages

Redescriptions are given of the mature oocysts of Isospora ameivae Carini, 1932, from the teiid lizard Ameiva ameiva, and Isospora hemidactyli Carini,1936 from the gecko Hemidactylus mabouia, in north Brazil. The endogenous stages of the two parasites in the small intestine are described. Those of I. ameivae are intracytoplasmic, whereas those of I. hemidactyli are intranuclear

Biology of Isospora spp. from humans, nonhuman primates, and domestic animals.

Coccidial parasites of the genus Isospora cause intestinal disease in several mammalian host species. These protozoal parasites have asexual and sexual stages within intestinal cells of their hosts and produce an environmentally resistant cyst stage, the oocyst. Infections are acquired by the ingestion of infective (sporulated) oocysts in contaminated food or water. Some species of mammalian Isospora have evolved the ability to use paratenic (transport) hosts. In these cases, infections can be acquired by ingestion of an infected paratenic host. Human intestinal isosporiasis is caused by Isospora belli. Symptoms of I. belli infection in immunocompetent patients include diarrhea, steatorrhea, headache, fever, malaise, abdominal pain, vomiting, dehydration, and weight loss, blood is not usually present in the feces. The disease is often chronic, with parasites present in the feces or biopsy specimens for several months to years. Recurrences are common, Symptoms are more severe in AIDS patients, with the diarrhea being more watery. Extraintestinal stages of I. belli have been observed in AIDS patients but not immunocompetent patients. Treatment of I. belli infection with trimethoprim-sulfamethoxazole usually results in a rapid clinical response. Maintenance treatment with trimethoprim-sulfamethoxazole is needed because relapses often occur once treatment is stopped.

Isolation of tissue cysts of Toxoplasma, Isospora, Hammondia and Sarcocystis from camel (Camelus dromedarius) meat in Saudi Arabia.

Meat samples were collected from the oesophagus and tongue of 38 camels slaughtered at the main abattoir of Al-Ahsa city, Saudi Arabia. Five cats and three dogs, conventionally reared and coccidia-free, were caged individually in steel cages. Camel meat was pooled, minced and fed to four cats and two dogs. One cat and one dog were not fed meat and were kept as noninfected controls. Faecal samples from infected and control animals were examined daily for a period of 2 months after feeding the meat. Three cats fed camel meat passed in their faeces oocysts of Toxoplasma gondii, Isospora felis and Isospora rivolta. The fourth cat passed only T. gondii and I. felis oocysts. One of the dogs fed camel meat passed oocysts of Isospora canis, Hammondia heydorni and Sarcocystis cameli sporocysts. The second dog excreted only S. cameli sporocysts.

Intestinal infection with Isospora belli

A review article discussing the taxonomy, parasite life cycle and laboratory diagnosis of Isospora belli is presented. The pathology, epidemiology, clinical findings and therapy of human intestinal infection with Isospora belli are also addressed

Isosporíase: atualizaçäo / Isosporiasis: review

Os autores fazem uma atualizaçäo sobre isosporíase humana e abordam os principais aspectos da parasitose: epidemiologia, quadro clínico, diagnóstico laboratorial e terapêutica clínica

[Isosporiasis and sarcocystosis. The current findings]. / Isosporiasi e sarcocistosi. Acquisizioni attuali.

A review on infections by Isospora belli and Sarcocystis spp. both in healthy and in AIDS patients is done on the basis of literature and personal data. In this view a special focus is made on isospora belli infection in AIDS because of its high recurrence after successful attack therapy. Consequently the most recent protocols for maintenance and attack therapy in these patients are reported. At the end, concerning ultrastructural pathology, the features of some Isospora belli developing stages are described by means of electron microscopy on duodenal biopsy specimens from a patient.

Recent advances in the knowledge of the biology of the cyst-forming coccidia.

A review is given on recent knowledge of the genera Caryospora, Isospora, Cystoisospora, Hammondia, Toxoplasma, Besnoitia, Sarcocystis and Frenkelia. Caryospora hitherto considered to be monoxenous was found to have an optional intermediate host. Many Isospora species had to be transferred to the genus Cystoisospora because optional intermediate hosts were discovered. The obligatory two-host genus Hammondia could be confirmed to be distinct from the genus Toxoplasma. Even though the life cycles of Besnoitia wallacei and B. darlingi could be elucidated, the mode of transmission of B. besnoiti which is of considerable economic importance is still unknown. Since the discovery of the life cycle of the sarcosporidia in 1972 more than 600 papers have been published on this parasite. At present, 122 Sarcocystis species are named, and of 56 species both the definitive and intermediate hosts are known. It was shown that not only carnivorous and omnivorous mammals but also birds of prey, owls, and reptiles are definitive hosts of Sarcocystis species. the close relationship between the genera Sarcocystis and Frenkelia was confirmed by several investigators. Biology and Sarcocystis are of special interest. In the developmental cycle of most Sarcocystis species there are two schizogonic generations in endothelial cells of blood vessels, and in some species there is an additional asexual multiplication by endodyogeny in white blood cells. Some Sarcocystis species are highly pathogenic in non-immune intermediate hosts. Moreover, the sarcocysts which hitherto have been considered to be apathogenic may impair their hosts. In horses, they may cause myopathy, and in pigs, they have considerable influence on several parameters determining meat quality.

Separation of Isospora (Toxoplasma) gondii cysts and cystozoites from mouse brain tissue by continuous density-gradient centrifugation.

A simple, quick and reproducible method consisting of density-gradient centrifugation of homogenized infected mouse brain tissue on Percoll is described for the isolation and purification of cysts of Isospora (Toxoplasma) gondii. A 100% recovery of cysts, with 74.2% in a single fraction with a specific gravity of 1.056, was obtained by overlaying homogenates of infected mouse brains on a pre-formed Percoll gradient and centrifugation at low g forces. With this procedure recovery was independent of the age of the cysts. Titration of purified cystozoites showed there to be no loss of infectivity.

[Comparative review of the developmental biology of the genera Sarcocystis, Frenkelia, Isospora, Cystoisospora, Hammondia, Toxoplasma and Besnoitia (author's transl)]. / Vergleichende Darstellung der Entwicklungsbiologie der Gattungen Sarcocystis, Frenkelia, Isospora, Cystoisospora, Hammondia, Toxoplasma und Besnoitia.

A review is given of the advances in our knowledge of the developmental biology of the so-called cyst-forming coccidia in the years from 1974 to 1978. Until 1970 only 6 Isospora species were known to occur in cats, dogs and men. After the discovery of the coccidian nature of the genera Toxoplasma, Sarcocystis, Besnoitia and Frenkelia, and after the discovery of the new genus Hammondia the number of known species rose to over 30. In addition it could be shown that also birds of prey, owls and reptiles serve as final hosts for several Sarcocystis and Frenkelia species. The coccidia with isosporoid oocysts can be classified into two major groups: Species with gamogony and sporogony in the final host (Sarcocystis, Frenkelia) and species with schizogony and gamogony in the final host and sporogony on the ground (Isospora, Cystoisospora, Hammondia, Toxoplasma, Besnoitia). The subdivision of the first group into the genera Sarcocystis and Frenkelia based on the localization of their cysts in the musculature and in the brain, respectively, cannot be upheld in the future. Their classification into organisms with small cystozoites of about 7 microm with birds or reptiles as final hosts (Sarcocystis and Frenkelia species of rodents) and those with large cystozoites of about 15 microm and mammals as final hosts (Sarcocystis spp. of domestic animals and rodents) would be more significative. The second group can be subdivided into monoxenous species (Isospora), species with an optional intermediate host in which no or only slight multiplication occurs (Cystoisospora) and in genera with a multiplication in two phases in the intermediate host (Hammondia, Toxoplasma, Besnoitia). The nomenclature of single species is very controversial. As an example the controversial apprehension of the taxonomy of the Sarcocystis species of cattle is discussed. An application has been submitted to the International Commission for the Zoological Nomenclature to delcare a number of names as nomina dubia and to introduce unambiguous names for those organisms for which type specimens are available.

Electron microscopy of stages of Isospora felis of the cat in the mesenteric lymph node of the mouse.

Stages of Isospora felis of the cat in the mesenteric lymph node of the mouse 25 days after oral inoculation with oocysts, have been described at the ultrastructural level. The organisms occurred singly within parasitophorous vacuoles in host cell cytoplasm and were sporozoite-like, having a large crystalloid body up to 5.5 mum in length posterior to the nucleus. The size and appearance of the parasitophorous vacuole varied. Some vacuoles contained numerous, small, electron dense granules about 30 nm in diameter. Because of the aggregation of granules and their arrangement within the parasitophorous vacuole, the impression was sometimes gained by light microscopy that parasites were surrounded by a sheath or cyst wall. However, a cyst wall was not present. In host cells, spherical, membrane-bound bodies with a homogeneous, electron dense core and a maximum diameter of 0.25 mum were filed along the limiting membrane of the parasitophorous vacuole. These extra-intestinal parasites were considered to be waiting stages, with a biological function similar to that of the tissue cyst stage of other general of isosporan coccidia.

[Light and electron microscope studies on cysts of Sarcocystis fusiformis in the muscles of calves infected experimentally with oocysts and sporocysts of the large form of Isospora bigemina from cats (author's transl)]. / Licht- und elektronenmikroskopische Untersuchungen an Cysten von Sarcocystis fusiformis in der Muskulatur von Kälbern nach experimenteller Infektion mit Oocysten und Sporocysten der grossen Form von Isospora bigemina der Katze

In several experiments young calves were infected with oocyss and sporocysts of the large form of isospora bigemina from cats, which has been fed by raw beef containing Sarcocystis fusiformis cysts. On he 98th and 160th day p.i. the calves were killed and the development of S. fusiformis cysts in muscle cells was sutided by lign and electron microscopy. The cyst was always situated within a muscle fiber which was never surrounded by fibrillar layers of host origin (= no secondary cyst wall). The cyst was limited by a singel unit membrane, which was thickened at numerous places of the interior by osmiophilic material. This complex is called primary wall (Primärhülle), reaching a thickness of up to 250 A. This primary wall was regulary folded, forming palisade-like protrusions...

[Light and electron microscopic studies on cysts of Sarcocystis fusiformis in the muscles of calves infected experimentally with oocytes and sporocysts of the large form of Isospora bigemina from dogs. 2. The fine structure of cyst stages].

In several experiments calves were infected with sporocytes of the large form of Isospora bigemina from dogs thus producing "thin-walled" cysts of Sarcocystis fusiformis in muscles. Following the growth of the cyst the development of the cyst stages (metrocytes, merozoites) was studied by electron microscopy. Cyst formation began about 34 days p.i. from a parasitophorus vacuole containing exclusively ovoid metrocytes. One the 62nd day p.i. mainly metrocytes and a few banana-shaped stages were present in the cysts. These banana-shaped stages were called merozoites, because we consider the process of cyst formation as an extraintestinal schizogony. From the 76th day p.i. mainly merozoites, i.e. infectious stages, were observed. The metrocytes were surrounded directly by the amorphous ground substance of the cyst's interior, whereas the merozoites were closely packed within chamber-like hollows of the ground substance. The metrocytes are globular cells, about 7 X 5 mum. These cells possess as the metrocytes of other species a typical three-layered pellicle with deep micropores, a conoid, polar ring with 22 anchored subpellicular microtubules, very few rhoptries and micronemes, a golgi complex anterior to the large nucleus. The nucleus has a spherical nucleolus consisting of granular and fibrillar zones. Chromosomal structures were seen in two different stages: large dense plaques (condensed stage), and as small dense granules of 300-400 A diameter, arranged spherically within the karyoplasm (extended stage). There is no significant difference in fine structure between the metrocytes of I. hominis-induced cysts and those from cysts after infections with the large form of I. bigemina from dog.

[Light and electron microscope studies on cysts of Sarcocystis fusiformis in the muscles of calves infected experimentally with oocysts and sporocysts of Isospora hominis Railliet et Lucet, 1891. 2. Fine structure of metrocytes and merozoites (author's transl)]. / Licht- und elektronenmikroskopische Untersuchungen an Cysten von Sarcocystis fusiformis in der Muskulatur von Kälbern nach experimenteller Infektion mit Oocysten und Sporocysten von Isospora hominis Railliet et Lucet, 1891. 2. Die Feinstruktur der Metrocyten und Merozoiten

In several experiments calves were infected with sporocysts of Isospora hominis thus producing "thick-walled" cysts of Sarcocystis fusiformis in muscles. Following the growth of the cyst the development of the cyst stages (metrocytes, merozoites) was studied by electron microscopy. Cyst formation began about 40 days p.i. from a parasitophorous vacuole containing exclusively ovoid metrocytes. On the 62nd day p.i. mainly metrocytes and a few banana-shaped merozoites were present within the cysts, whereas on the 98th day p.i. only merozoites, i.e. infectious stages, were observed. The metrocytes were surrounded directly by the amorphous ground substance of the cyst's interior, but the merozoites were arranged relatively loose within chamber-like hollows of the ground substance. The metrocytes are globular cells, about 6-7 mum by 4. 5 mum. The typical three-layered pellicle had a few invaginations and several micropores, which seem to ingest numerous small vesicles from the interior of the cyst. These cells posses a conoid, polar ring with 22 anchored supellicular microtubules, several rhoptries and micronemes, a glogi complex anterior to the large nucleus. The nucleus has a spherical nucleolus consisting of granular and fibrillar zones. Chromosomal structres were seen in two different stages: large dense plaques (condensed stage), and as small dense granules of 300-400 a diameter, arranged sperically within the karyoplasm ( extended stage). The nuclear pores show the typical eight-fold symmetry known from other protozoa and numerous metazoa. The rought endoplasmic reticulum is very prominent within these cells as well as the tubular mitochondria...

[Light and electron microscope studies on cysts of Sarcocystis fusiformis in the muscles of calves infected experimentally with oocysts and sporocysts of the large form of Isospora bigemina from dogs. 1. The development of cysts and "cyst wall" (author's transl)]. / Licht- und elektronenmikroskopische Untersuchungen an Cysten von Sarcocystis fusiformis in der Muskulatur von Kälbern nach experimenteller Infiktion mit Oocysten und Sporocysten der grobetaen Form von Isospora bigemina des Hundes. 1. Zur Entstehung der Cyste und der "cystenwand"

In several experiments young calves were infected with Isospora bigemina (large form) sporocysts excreted by dogs which had been fed with raw beef containing Sarcocystis fusiformis cysts. On the 27th, 34th, 62nd, 76th and 150th day p.i. the calves were killed and the development of S. fusiformis cysts in muscles cells was studied by light and electron microscopy. On the 27th day p.i. in light microscope preparations numerous schizonts, merozoites and endodyogeny-stages were seen in various organs, such as the liver, lung, kidney, heart, small intestine, esophagus, skeletal muscles, diaphragm, cerebrum, and cerebellum. The merozoites measured 7-8 mug by 2-3 mum. Beginning with the 34th day p.i. numerous cysts containing small numbers of metrocytes only were observed in electron microscopy, too. The cysts developed from a parasitophorous vacuole within the host cells. At first this vacuole was limited by a single unit membrane, which soon became thickened byosmiophilic material at numerous places inside of the vacuole. This complex, called primary wall (= Primärhülle), reached a thickness of up to 200-250 A in all cysts. During growth of the cyst this primary wall became regularly folded forming alternating long and short club-shaped protrusions. The longer protrusions were about 0.6 mum long and 0.2-0.3 mum in diameter, whereas the short protrusions were of about 0.13 mum in length. In light microscopy the combined protrusions had the appearance of a very thin cyst wall because of their small size and their close proximity to each other. Later, all protrusions became longer with a maximum of about 3 mum in length without any change in the diameter. Yet, from the 76th day p.i. these protrusions appeared no longer straight, but they became folded over, following a course along the surface of the cyst. Evidently the protrusions did not increase in number after their initial formation, for the distance between them became greater in older cysts. No fibrillar elements were seen within these protrusions which probably accounts for the folding over. The zone of the superficial folded protrusions was not thicker than 1 mum so that in light microscope even the old cysts appeared as relatively thin walled. The interior of the original electron-pale parasitophorous vacuole bacame progressively condensed during the growth of the cyst. There was development of an amorphous ground substance, containing fine fibrils and granules. The ground substance became divided into thin speta (not visible with the light microscope) forming numerous changer-like hollows. The parasites were very closely packed within these hollows. At the beginning of the cyst formation only metrocytes were found within the young cysts, whereas on the 76th day p.i. and later only the infectious merozoites were present. It is therefore concluded that about 3 months after inoculation of calves with sporocysts of the large form I. bigemina from dogs the cysts are fully differentiated, thus being ready for a new transmission...