Outer Oocyst Wall Research Articles

Distribution and Processing of Eimeria nieschulzi OWP13, a New Protein of the COWP Family.

Eimeria species are important veterinary coccidian parasites and are transmitted between hosts via oocysts. The infectious sporozoites are protected by the oocyst and sporocyst wall. Tyrosine-rich proteins are well-known components of the Eimeria oocyst wall. Recently, cysteine motif containing proteins (COWP family), as described in Toxoplasma gondii and Cryptosporidium spp., have also been characterized in Eimeria. Here, we identified a novel COWP-related protein, EnOWP13, and tracked it via transfection technology in Eimeria nieschulzi. The subsequent analysis suggests that the mCherry-tagged EnOWP13 localizes to the wall-forming bodies I and the outer wall. Immunohistochemical analysis confirmed the distribution of wall-forming bodies similar to avian Eimeria species and revealed that the wall-forming bodies I show peroxidase activity. The EnOWP13 amino acid composition and FITC-cadaverine-positive wall-forming bodies I suggest a participation of an enzyme with transglutaminase activity. This is the first description and characterization of this novel outer oocyst wall protein, which is also orthologous to other Eimeria species and Toxoplasma gondii, suggesting a new potential cross-linking mechanism of wall-forming proteins via isopeptide bonds.

F-actin distribution and function during sexual development in Eimeria maxima.

To determine the involvement of the actin cytoskeleton in macrogametocyte growth and oocyst wall formation, freshly purified macrogametocytes and oocysts were stained with Oregon Green 514 conjugated phalloidin to visualize F-actin microfilaments, while Evans blue staining was used to detect type 1 wall forming bodies (WFB1s) and the outer oocyst wall. The double-labelled parasites were then analysed at various stages of sexual development using three-dimensional confocal microscopy. The results showed F-actin filaments were distributed throughout the entire cytoplasm of mature Eimeria maxima macrogametocytes forming a web-like meshwork of actin filaments linking the type 1 WFBs together into structures resembling 'beads on a string'. At the early stages of oocyst wall formation, F-actin localization changed in alignment with the egg-shaped morphology of the forming oocysts with F-actin microfilaments making direct contact with the WFB1s. In tissue oocysts, the labelled actin cytoskeleton was situated underneath the forming outer layer of the oocyst wall. Treatment of macrogametocytes in vitro with the actin depolymerizing agents, Cytochalasin D and Latrunculin, led to a reduction in the numbers of mature WFB1s in the cytoplasm of the developing macrogametocytes, indicating that the actin plays an important role in WFB1 transport and oocyst wall formation in E. maxima.

Antigen incorporation on Cryptosporidium parvum oocyst walls.

Cryptosporidium parvum oocysts are the infective stages responsible for transmission and survival of the organism in the environment. In the present work we show that the oocyst wall, far from being a static structure, is able to incorporate antigens by a mechanism involving vesicle fusion with the wall, and the incorporation of the antigen to the outer oocyst wall. Using immunoelectron microscopy we show that the antigen recognized by a monoclonal antibody used for diagnosis of cryptosporidiosis (Merifluor(R), Meridian Diagnostic Inc.) could be found associated with vesicles in the space between the sporozoites and the oocysts wall, and incorporated to the outer oocyst wall by an unknown mechanism.

Development of a mathematical model of Cryptosporidium inactivation by ozonation

A new mathematical model was developed to express the processes of Cryptosporidium inactivation by ozonation. In this model, five different stages were considered as state variables of Cryptosporidium oocysts for accurate expression of the inactivation. ATP, in vitro excystation and DAPI/PI permeability assays were used to describe the oocyst amounts of different stages. Some reaction constants were estimated by the structure components of oocysts or by the stoichiometry of reactions, while the others were by the oocysts population changes in ozonation. The calculated values of this model were well consistent with experimental inactivation data. Three-log inactivation of sporozoites required about 0.04 mgO3 per unit oocyst (mgC) from the simulation results. Before ozone reacts with sporozoites, more ozone was consumed to oxidize other parts of oocysts and DOC produced. The main path of inactivation of oocysts by ozonation was estimated to be P1 (intact oocysts)→P2 (oocysts with damaged outer oocyst wall)→P4 (oocysts without excystation function)→P5 (oocysts with inactivated sporozoites and no excystation function) from experimental and simulated results.

A comparative study on the biology of Cryptosporidium sp. from guinea pigs and Cryptosporidium parvum (Apicomplexa)

Cryptosporidium sp. from guinea pigs and C. parvum were compared morphologically, electrophoretically, and for the ability to infect suckling mice. Oocysts from guinea pigs measured 5.4 x 4.6 (4.8-5.6 x 4.0-5.0) microns and had a shape index (length/width) of 1.17 (1.04-1.33). Oocysts of C. parvum were similar and measured 5.2 x 4.6 (4.8-5.6 x 4.2-4.8) microns with a shape index of 1.16 (1.04-1.33). All suckling mice inoculated with oocysts of C. parvum became infected, whereas most, but not all, mice fed oocysts of the guinea pig isolate also became infected. However, mice inoculated with oocysts from guinea pigs produced on average 100-fold fewer oocysts by day 7 postinoculation than did mice infected with C. parvum, and the resulting infections were sparse and patchy along the ileum. Electrophoretic profiles were similar, but 125I surface labeling of outer oocyst wall proteins revealed striking differences between the two isolates. Cryptosporidium parvum had a wide molecular size range of 125I-labeled bands, whereas C. sp. from guinea pigs had a banding pattern clustered between 39 and 66 kDa, with a smaller number of bands greater than 100 kDa.

Identification of outer oocyst wall proteins of three Cryptosporidium (Apicomplexa: Cryptosporidiidae) species by 125I surface labeling

Autoradiography of oocyst wall surface proteins of three Cryptosporidium spp. revealed common bands at 285 to 290, 145 to 148, 120, 57, and 32 kilodaltons (kDa). Cryptosporidium baileyi and C. muris share proteins at 180, 100, 80 to 81, 29, and 18 to 19 kDa; C. baileyi and C. parvum share one protein at 46 to 47 kDa; and C. muris and C. parvum share a protein at 67 to 69 kDa. Additional protein bands, each unique to one species, were also observed.

Eimeria from Bats of the World. II. A New Species in Tadarida femorosacca from Sonora, Mexico

Between 1979 and 1980, 104 bats representing 13 species in 4 families were collected in California and New Mexico, U.S.A., and Baja California and Sonora, Mexico, and were examined for coccidia; only 3 (3%) had oocysts in their feces. Bats examined and their infection rates were: Molossidae: 0 of 12 Tadarida brasiliensis, 1 of 18 (6%) T. femorosacca; Natalidae: 0 of 1 Natalus stramineus; Phyllostomatidae: 0 of 1 Choeronycteris mexicana, 0 of 2 Leptonycteris sanborni, 0 of 1 Macrotus californicus; Vespertilionidae: 0 of 9 Antrozous pallidus, 0 of 28 Eptesicus fuscus, 0 of 1 Lasionycteris noctivagans, 0 of 3 Lasiurus borealis, 2 of 22 (9%) L. cinereus, 0 of 1 L. ega, 0 of 5 Pipistrellus hesperus. Sporulated oocysts were only found in T. femorosacca and these represent a new species, Eimeria tadarida n. sp. They are subspheroidal to ellipsoidal, 19 x 25 (16-23 x 20-30) microns; a micropyle is absent, and fragments within the oocyst may be oocyst residuum or multiple polar bodies. The oocyst wall, approximately 1.5 microns, is composed of a mammillated outer layer and smooth inner layer. Sporocysts are ovoidal, 8 x 12 (6-9 x 10-14) microns, and have a small Stieda body and a wide substieda body. This is only the 14th eimerian to be described from bats worldwide. Only unsporulated or partially sporulated oocysts of an eimerian were seen in 2 L. cinereus. These measured 28 x 25 (27-29 x 24-26) microns and had a mammillated outer oocyst wall.

Coccidian parasites (Apicomplexa: Eimeriidae) from insectivores. II. Six new species from Japanese shrew moles (Talpidae).

Thirty-eight of 51 (74.5%) shrew moles collected in Japan were infected with from one to four species of Eimeria and/or Isospora including six of six Dymecodon pilirostris and 32 of 45 (71.1%) Urotrichus talpoides. Four eimerians and two isosporans were identified and all are described as new species. Sporulated oocysts of Eimeria amorphospora n. sp. were subspheroid/ellipsoid, 21.1 x 17.9 (18-25 x 16-21) micrometers. Sporocysts were amorphous, gelatinoid envelopes greater than or equal to 20.3 x 7.5 (17-24 x 7-9) micrometers. Sporozoites were enclosed together within a membrane in each sporocyst. This species was found in 9 of 45 (20%) U. talpoides. Sporulated oocysts of Eimeria gonocilia n. sp. were subspheroid/ellipsoid, 28.8 x 24.4 (25-30 x 21-28) micrometers; a highly ornate outer oocyst wall gave the appearance of a ciliated ball. Sporocysts ovoid, pointed at both ends, were 17.0 x 9.9 (15-19 x 7-11) micrometers; this species was found in 4 of 45 (8.9%) U. talpoides. Sporulated oocysts of Eimeria talpoidei n. sp. were asymmetrical ovoid, 20.6 x 13.3 (18-23 x 12-15) micrometers, with sporocysts lacrimiform, 12.0 x 5.8 (10-14 x 5-7) micrometers. This species was found in 7 of 45 (15.6%) U. talpoides. Sporulated oocysts of Eimeria honshuensis n. sp. were ellipsoid, 15.5 x 11.4 (13-18 x 10-13) micrometers, with sporocysts ovoid, 9.1 x 5.2 (8-10 x 4-6) micrometers. This species was found in 10 of 45 (22.2%) U. talpoides and in 5 of 6 (83.3%) D. pilirostris. sporulated oocysts of Isospora dymecodi n. sp. were subspheroid/ellipsoid, 15.8 x 12.6 (13-17 x 11-13) micrometers, with sporocysts ovoid, 9.1 x 5.2 (8-10 x 4-6) micrometers. This species was found in 10 of 45 (22.2%) U. talpoides and in 5 of 6 (83.3%) D. pilirostris. Sporulated oocysts of Isospora dymecodi n. sp. were subspheroid/ellipsoid, 15.8 x 12.6 (13-17 x 11-13) micrometers, with sporocysts ellipsoid, 10.9 x 6.9 (10-13 x 6-8). This species was found in six of six D. pilirostris. Sporulated oocysts of Isospora urotrichi n. sp. were spheroid/subspheroid, 13.4 x 12.4 (11-16 x 9-14) micrometers, with sporocysts ovoid, 9.2 x 6.3 (8-11 x 5-7) micrometers. This species was found in 27 of 45 (60%) U. Talpoides. Only 14 of 38 (36.8%) infected hosts (one D. pilirostris, 13 U. talpoides) were seen to be naturally infected with only one coccidian species when sampled.

Coccidiosis of sandhill cranes (Grus canadensis) wintering in New Mexico.

The feces of 212 sandhill cranes (Grus canadensis) collected in central New Mexico from October 1982 to January 1983 and from October 1983 to January 1984 were examined to determine the prevalence of coccidial oocysts. One hundred forty-five granulomatous nodules from the viscera of 64 cranes and samples of lung, small intestine, and large intestine from 58 birds were examined by light and transmission electron microscopy for the presence of intestinal or extraintestinal coccidiosis. Of the 212 fecal samples, 160 (75%) were positive for oocysts of Eimeria, including E. gruis in 139 (66%) and E. reichenowi in 118 (56%) of the samples. Eimeria bosquei sp. n. was found in two (approximately 1%) of the fecal samples. Subspheroid to ovoid oocysts of this new species are 19-27 X 14-19 (23.6 X 17.1) micrograms with ovoid sporocysts 10-14 X 7-11 (12.3 X 9.3) micrograms. A rough, heavily pitted outer oocyst wall, sporocyst residuum, Stieda and substieda bodies, and multiple polar bodies are present. The polar bodies, of varying sizes, always aggregate at the apex of the sporulated oocyst. An Adelina sp. was found in one (0.5%) crane. Coccidian developmental stages were found in the epithelium and lamina propria of the small and large intestine. Disseminated granulomatous nodules were found in the oral mucosa, esophagus, heart, descending aorta, liver, small intestine, mesenteries, and parietal peritoneum. Unique cell types resembling coccidian asexual and sexual stages were observed by light and electron microscopy in some of the nodules.

Video Microscopy of Swimming and Secreting Cercariae of Schistosoma mansoni

termined with accuracy. However, E. monacis and E. perforoides oocysts sporulated first with about 85% sporulating after 5 days incubation while E. os took a slightly longer time (about 6.5 days). Eimeria tuscarorensis required about 20 days for a 50% sporulation. A short sporulation time may be a factor for the relative abundance of E. monacis and E. perforoides. Depending upon the time of year, between 77% and 90% of the oocysts were E. monacis and E. perforoides. In contrast, sporulation in E. tuscarorensis required at least 4 times longer than E. monacis and E. perforoides. The relatively long sporulation period would require more time for E. tuscarorensis to achieve sufficient levels of infective oocysts in the environment to produce an increase in oocyst numbers in the host. Furthermore, E. tuscarorensis oocysts were relatively more prevalent during the warmdry summer weather months (May-August) suggesting that the thick outer oocyst wall may resist desiccation and extend viability. Eimeria os were more prevalent in the Spring and Fall, suggesting less resistance to desiccation or heat. Indeed, E. os oocyst appeared to deteriorate much sooner than other species when incubated at 28-30 C in 2.5% potassium dichromate. The deterioration usually started at the region around the micropyle. In conclusion, coccidial parasites were abundant in the woodchuck population of southern Illinois. Although E. monacis and E. perforoides were the species which predominated through the non-hibernating months of the year, E. os and E. tuscarorensis appeared to show seasonal

Eimeria dalli sp. n. (Protozoa: Eimeriidae) from Dall sheep Ovis dalli.

SYNOPSISEimeria dalli sp. n. is described from fecal samples collected from Dall sheep, Ovis dalli, from the Kenai Peninsula, Alaska. The oocysts are spherical or subspherical with mean dimensions of 43.7 × 37.4 μm. The outer oocyst wall is rough and irregular. No micropyle, micropylar cap or residuum was observed. Sporocysts were elongate ovoid with mean dimensions of 19.0 × 10.7 μm. Stieda bodies were not discernible.

Eimeria ovata n. sp. and Other Coccidia of the Eastern Chipmunk Tamias striatus in Massachusetts

SYNOPSIS. Eimeria vilasi, E. wisconsinensis, and E. ovata n. sp. were found in the intestinal contents of eastern chipmunks Tamias striatus in Massachusetts. The oocysts of E. ovata are ovoid with mean dimensions of 26.1 × 16.7 μ. The outer oocyst wall is rough, with small pits. Micropyle and oocyst residuum are absent. Sporulation time at room temperature is 7–8 days.