The third newly discovered Eimeria species (Protozoa: Eimeriidae) described from wild reindeer, Rangifer tarandus, in Iceland

Fecal samples from 56 adult reindeer from eastern Iceland were examined for coccidian parasites. One Eimeria species was found in an 8-year-old male. Prevalence of infection was 1.8%; oocyst per gram (opg) value was 150. The coccidium was identified and described as a new species. The sporulated oocysts are ellipsoidal and average size is 30.0×21.1 μm. The oocyst has two distinct walls. Wall thickness is ~1.0 μm, and the outer wall, ~four-fifths of total thickness, is generally smooth and appears bicoloured. The outermost portion is light blue, and the innermost portion, yellow to pale brown. The inner wall is dark brown. Oocysts contain a prominent polar granule but are devoid of a micropyle. Oocysts enclose four spindle-shaped sporocysts with a rounded end opposite to the Stieda body. The average size of sporocysts is 15.3×6.5 μm. Sporocysts contain a granular sporocyst residuum that usually forms a cluster between the sporozoites and one large refractile body in each sporozoite.     

Utrastructural observations on <Emphasis Type="Italic">Goussia metchnikovi</Emphasis> (Laveran, 1897) in the spleen of gudgeon, <Emphasis Type="Italic">Gobio gobio</Emphasis> L.

The ultrastructure of gamonts and sporulated oocysts of Goussia metchnikovi in the spleen of gudgeon, Gobio gobio from the river Lee, England is described. In developing microgamonts, small amylopectin granules were grouped centrally and nuclei were often arranged peripherally, close to the surface membrane. Nuclear chromatin condensed into peripheral dense portions that became the nuclei of flagellated microgametes, released to the parasitophorous vacuole. The cytoplasm of macrogametes had larger, scattered amylopectin granules, lipid globules and small electron-dense bodies, but no obvious wall forming bodies; peripheral vesicular structures with the appearance of mitochondria were also present and the parasitophorous vacuole contained flocculent material, but was otherwise free of structures. Sporulated oocysts contained four sporocysts and oocyst walls appeared to consist of a single membrane. Sporocyst walls showed a dehiscence suture, characteristic of the genus Goussia, which had filamentous extensions in places. The sporocyst wall comprised a dense inner layer and a thin outer layer with a fuzzy coat, separated by an electron lucent layer. Groups of oocysts were encapsulated by fibrous layers and inflammatory cells, and many sporocysts and their contained sporozoites showed evidence of elimination by the host.     

Excystation ofIsospora suis Biester, 1934 of swine

The in vitro excystation of sporozoites ofIsospora suis Biester 1934 is described. Sporocysts ofI. suis lack a Stieda body. Upon incubation in 0.75% sodium taurocholate or in 0.25% trypsin+0.75% sodium taurocholate excystation solutions, sporozoites were released by separation of the sporocyst wall into four plates. Occasionally, the sporocyst wall did not separate completely but opened partially and released the sporozoite. At the time of excystation, sporozoites were short and broad but became elongated after 5 to 10 min in the excystation fluids. Elongate sporozoites measuring 11.7×3.8 m, had a pointed anterior end and a nucleus located in the posterior half of the cell. Living sporozoites exhibited gliding movements, side-to-side flexion, and probed with their anterior ends. Incubation in 5.25% sodium hypochlorite removed the oocyst walls from most oocysts. Sporozoites did not excyst from sporocysts that were released during treatment with sodium hypochlorite.     

New exogenous stages of oocysts,sporocysts, and sporozoites of <Emphasis Type="Italic">Goussia cichlidarum</Emphasis> Landsberg and Paperna 1985 (Sporozoa: Coccidia) and impact of endogenous stages on the swim bladder of tilapias in Egypt

Obviously, the present study reports the coccidian parasite so-called Goussia cichlidarum for the first time in Egypt. Altogether, 25 exogenous stages were clearly distinguished from specimens of naturally infected fishes of Oreochromis niloticus, O. auraeus, and Tilapia zillii from different locations. The total prevalence of infection was about 41%. Mostly, infected fish grossly seemed with a healthy body, although severe lesions have been detected microscopically in massive infection. Portions of thick wall swim bladder have been placed in vitro. The released parasitic stages have been photographed, sketched, measured, described, and compared with previously described species. Oocysts, sporocysts, and sporozoites have also been differentiated morphologically and morphometrically. Maturity stages of sporozoites containing sporocysts within either an oocyst or those released and sporulated outside the oocyst were considerably discernible. In addition, endogenous stages have also been investigated in histological sections included gamonts, merozoites, oocysts, and different stages of sporozoites.     

The sporulation ofEimeria tenella as revealed by a novel preparative method

A simple technique for obtaining ultra-thin sections of coccidian oocysts is reported. Oocyst walls, the main barriers to fixatives, dehydrating agents and embedding media are ruptured by brief immersion of the organism in liquid nitrogen. Adequate penetration of chemicals is thus assured but with minimal damage to the internal tissue. The fine structure of sporulation of oocysts ofEimeria tenella was studied using this process. Each oocyst produced four sporoblasts. These developed into sporocysts and each produced two sporozoites.     

Scanning electron microscopy of poultry coccidia after in vitro excystation and penetration of cultured cells

Summary The surface structure of Eimeria tenella and E. adenoeides oocysts, sporocysts, and sporozoites were studied by scanning electron microscopy to determine if changes in these surfaces occurred during in vitro excystation and following penetration into cells cultured in monolayers. Oocysts have a rough textured proteinaceous coat. Surface sterilization with NaOCl removed the outer covering of the oocysts, leaving a smooth surface. Sporocysts released from ruptured oocysts had a smooth textured surface. The sporocyst hull covered all of the Stieda body except the tip which was located partly outside the narrow end of the sporocyst. The opening of the sporocyst was slightly ruffled. Excysted sporozoites had a rough surface with small pits and grooves suggestive of micropores and other pellicular invaginations and constrictions. The anterior end of the sporozoites possessed a well defined truncated protrusion of the conoidal complex, the rostrum. We attribute this phenomenon to the activation of the sporozoite by the trypsin-bile solution.A portion of this paper was presented at the 44th Annual Meeting of the American Society of Parasitologists, November 3–7, 1969, Washington, D.C., U.S.A. Abbreviations for all figures: A Polysaccharide granules, CR Rostrum—protruded conoidal apparatus, M P Micropore, Oh Oocyst hull, R B Refractile body, S B Stieda body, Sc Sporocyst, Sc R Sporocyst residuum, Sh Sporocyst hull, Sz SporozoiteFig. 1. Freeze dried oocyst of E. tenella from which the outer proteinaceous layer has been removeed by surface sterilization with 5% NaOCl. The surface is smooth and no micropyle is visible. Notice the bacteria adjacent to the oocyst (arrow). These were either contaminants from the specimen peg or from the oocyst culture which had been killed by the NaOCl, but not laysed. Cell cultures inoculated with the same material had no bacterial growth. ×3750Because of the depth of focus and tilt of the stage, the indicated magnifications apply only to the center of the field and are, therefore, nominal magnifications.Fig. 2. Higher magnification of a surface sterilized oocyst. The thin inner layer of the oocyst hull has collapsed during preparation. Some venation is apparent (arrow). ×10000Fig. 3. The surface of an E. tenella oocyst which was not sterilized with NaOCl. The rough proteinaceous outer layer is still intact. Some debris (*) has settled onto the oocyst during preparation. ×10000     

The kinetics of oocyst shedding and sporulation in two immunologically distinct strains of Eimeria maxima, GS and M6

The kinetics of oocyst shedding and sporulation of two immunologically distinct strains of Eimeria maxima (GS and M6) were compared. Both strains had a prepatent period of approximately 120?h followed by peak oocyst shedding at 144-150?h post inoculation. Mean total oocyst output determined for each strain demonstrated that the fecundity of the M6 strain (12.8?×?10³?±?1.95) of E. maxima was roughly twice that of the GS strain (6.9?×?10³?±?3.33) when inoculated at the rate of 1,000 infective oocysts per bird. The process of oocyst sporulation was followed by repetitive sampling of sporulating oocysts at 26?°C with aeration over a 138 hour period. Sporulation was divided into five morphologically distinguishable stages whose abundance peaked at the following times during sporulation: unsporulated oocysts at 0?h; sporoblast anlagen at 18?h; sporoblasts without sporocyst walls at 22?h; and sporocysts without mature sporozoites at 38?h. The time to 50?% sporulation of E. maxima oocysts observed in the present study was approximately 53?h for both strains and all viable oocysts had completed sporulation by 60?h. In the present study, the prepatent periods, duration of oocyst shedding, and the relative kinetics of sporulation of the GS and M6 strains of E. maxima were found to be virtually identical despite the immunological distinctiveness of these two parasite strains.     

Electron microscopic study on sporocysts and sporozoites of parental strains and precocious lines of rabbit coccidia Eimeria intestinalis, E. media and E. magna

The fine structure of sporocysts and sporozoites of parent strains and precocious lines of rabbit coccidia Eimeria intestinalis, E. magna and E. media was studied. The parent strains and precocious lines differ only in the shape and size of refractile bodies (RB). In the sporocysts of precocious lines of E. magna and E. media, one extremely large RB was seen, either inside one of the sporozoites, or free in the sporocyst. In the oocysts of the precocious strain of E. intestinalis, two sporocysts resembled those of the precocious lines of E. magna and E. media, whereas the other two sporocysts did not harbour any RB. Sporozoites of all the precocious lines contained no, or very small, RB after in vitro excystation. Received: 28 July 2000 / Accepted: 4 August 2000     

Localization and immunogenicity of a low molecular weight antigen of Eimeria tenella

A low molecular weight (LMW) antigen recognized by a murine monoclonal antibody (C₃4F₁) was localized within endogenous stages of Eimeria tenella (USDA strain 80). Using indirect fluorescent antibody assay and immunoelectron microscopy, the LMW antigen was found in: sporozoites, first, second and third generation meronts, gamonts, unsporulated oocysts, and sporocysts. The antigen was observed in the cytoplasm and pellicle of the parasite, and in the parasitophorous vacuole, sporocyst walls and cytoplasm of infected host cells. The immunogenicity of this LMW antigen was assessed by antigen-specific serum antibody responses in chickens orally inoculated with live oocysts or injected intramuscularly with dead sporozoites. LMW antigen-specific serum antibodies were detected using Western blots of E. tenella sporozoites as early as 4 days after sporozoite injection and 6 days after oocyst inoculation. Unusually, the monoclonal antibody C₃4F₁ reduced the binding of immune chicken serum to the antigen in a competitive antibody binding assay, but not the reverse, suggesting that there is a single, immunodominant epitope on this antigen.     

Eimeria separata: method for the excystation of sporozoites

A method is described for the excystation and collection of infective sporozoites of Eimeria separata. The procedure uses conditions that resemble the in vivo environment. The first treatment of the oocysts in a 0.4% pepsin/HCl solution alters the oocyst wall, which becomes thinner. The second treatment in a 0.4% trypsin/0.75% taurocholate solution breaks the oocyst wall and sporocysts are released. A third incubation of the oocyst-sporocyst mixture in trypsin-free medium with 0.75% taurocholate and an additive of MgCl₂ followed by a final incubation in RPMI medium supplemented with 1% fetal calf serum yields a sporozoite excystation rate of up to 90%. Received: 23 October 1998 / Accepted: 17 November 1998     

Transformation of oocysts from several coccidian species by heat treatment

The transformation of sporocysts in oocysts ofEimeria caviae, E. intestinalis, E. nieschulzi, E. separata, E. tenella, Isospora canis, I. heydorni, andToxoplasma gondii by heat treatment was examined. Fresh unsporulated oocysts from these species were heated at 50° or 55°C for less than 5 min and cultivated at 25°C for 1 week. No transformation of sporocysts was observed in the sporulated oocysts following heating at 55°C, but sporulated oocysts ofTyzzeria andCaryospora types were found in someI. canis andI. heydorni oocysts after heating at 50°C followed by cultivation. A few sporulated oocysts of theIsospora type were observed inE. intestinalis oocysts after cultivation. All transformed sporulated oocysts contained eight sporozoites. In contrast, the number of sporocysts varied from two to 0. These results indicate that sporocyst formation in oocysts was affected by heat treatment. The relationship between the total numbers of sporozoites and sporocysts in the transformed oocysts was consistent with that of the Octozoic group classified by Hoare.     

New isosporoid coccidian parasites of sayaca tanager, Thraupis sayaca, from South America

Three new coccidian (Apicomplexa, Eimeriidae) species from the sayaca tanager, Thraupis sayaca, from Brazil, are reported in the current study. Isospora sanhaci sp. nov. oocysts are spherical to subspherical, 22.1 × 21.0 μm, with smooth, bilayered wall, ∼1.0 μm. Micropyle, oocyst residuum and polar granule are absent, while Stieda and substieda bodies are present. Sporocysts are elongated, 17.0 × 9.9 μm. Sporocyst residuum is present, sporozoites have one refractile body and a nucleus. Isospora sayacae sp. nov. are spherical to subspherical, 28.9 × 27.4 μm, with smooth, bilayered wall, ∼1.3 μm. Micropyle, oocyst residuum and polar granule are absent, while Stieda and substieda bodies are present. Sporocysts are bottle-shaped, 23.4 × 11.8 μm and containing a sporocyst residuum and sporozoites with one refractile body and a nucleus. Isospora silvasouzai sp. nov. are spherical to subspherical, 25.5 × 22.6 μm, with a smooth, bilayered wall, ∼1.0 μm. Micropyle and oocyst residuum are absent, but one polar granule is present. Sporocysts are pyriform ellipsoidal, 17.6 × 10.5 μm. Stieda and substieda bodies and sporocyst residuum are present and sporozoites have one refractile body and a nucleus.     

Fine structure of the oocyst wall and excystation ofEimeria procyonis from the American raccoon (Procyon lotor)

Oocysts ofEimeria procyonis, from the American raccoon (Procyon lotor), were broken, added to a cell suspension, fixed in Karnovsky's fluid, and studied with the electron microscope. The oocyst wall has three layers: a thin electron-dense inner layer (8–15 nm), an electron-lucent middle layer (25–35 nm), and a thick outer layer (120–140 nm). The outer layer has an electron-dense inner portion and an electron-lucent outer portion that contains membrane-bound vesicles. When exposed to a trypsin-sodium taurocholate fluid, sporozoites excysted from most sporocysts which were 35–43 months old, but not from sporocysts that looked normal and were 106 months old. Excysted sporozoites measured 13–16×3–4 (mean 14.3×3.2) m, usually had two refractile bodies, and had a nucleus with a prominent nucleolus.     

Characterization and immunoprotective properties of a monoclonal antibody against the major oocyst wall protein of Eimeria tenella.

The oocyst wall of Eimeria spp. consists of a 10-nm-thick outer lipid layer and a 90-mm-thick inner layer of glycoprotein which has been described previously to be composed of a single major protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and (125)I labelling of a oocyst wall fragments and of delipidated intact oocysts revealed a molecule of approximately 12 kDa as the major protein component of the oocyst wall of Eimeria tenella. An immunoglobulin M monoclonal antibody (c11B9F3) was produced against this 12-kDa oocyst wall protein sliced from a preparative SDS-polyacrylamide gel. Its reactivity by immunofluorescence against oocyst wall fragments and sporozoites or by immunoperoxidase assays of infected tissue sections was stage restricted to gametocytes and oocysts but pan-specific against all face of the oocyst wall. In chicks passively immunized with C11B9F3, oocyst output was significantly (P<0.01) reduced by 42 to 54% after homologous E. tenella infection and by 35% after heterologous Eimeria maxima infection compared with that of control groups. The results demonstrate the presence of a highly conserved, low-molecular-weight antigen on the oocyst wall and the gametocytes of Eimeria spp. which is a candidate for inclusion in a pan-specific, transmission-blocking vaccine against avian coccidiosis.     

Purification of<Emphasis Type="Italic"> Sarcocystis neurona</Emphasis> sporocysts from opossum (<Emphasis Type="Italic">Didelphis virginiana)</Emphasis> using potassium bromide discontinuous density gradient centrifugation

This report describes a new, inexpensive procedure for the rapid and efficient purification of Sarcocystis neurona sporocysts from opossum small intestine. S. neurona sporocysts were purified using a discontinuous potassium bromide density gradient. The procedure provides a source of sporocyst wall and sporozoites required for reliable biochemical characterization and for immunological studies directed at characterizing antigens responsible for immunological responses by the host. The examined isolates were identified as S. neurona using random amplified polymorphic DNA primers and restriction endonuclease digestion assays. This method allows the collection of large numbers of highly purified S. neurona sporocysts without loss of sporocyst viability as indicated by propidium iodide permeability and cell culture infectivity assays. In addition, this technique might also be used for sporocyst purification of other Sarcocystis spp.     

Fine structure of sporogonic stages of Goussia clupearum (Apicomplexa: Eimeriidae) in the liver of infected fish (Belone belone L.), using light and electron microscopy

A coccidian species, Goussia clupearum (L.) is reported to parasitize the liver of a new host, Belone belone (Teleostei: Belonidae), caught on the Atlantic coast at the north of Portugal. The parasitophorous vacuole containing oocysts was attached to the host's liver cells. Spherical oocysts (∼ 21.2 μm diameter), each containing four ellipsoidal elongated sporocysts (10.5 × 6.3 μm), were enclosed in the parasitophorous vacuole. Each sporocyst contained two sporozoites. The micropyle was absent, but a polar granule (without Stieda body) was present. Each sporozoite possessed four refractile bodies. During sporoblastogenesis and sporogenesis, one or two dense polar bodies were found within the oocysts. They were composed of a dense homogeneous core, surrounded by a ring of dense granular material. On occasion, we observed some sporocysts in direct contact with host cells. This paper describes the morphology and ultrastructural details of the oocysts, sporocysts and sporozoites of G. clupearum. This species seems to represent the only coccidium described in fish from this Atlantic coast. Received: 5 August 2000 / Accepted: 12 October 2000     

Ultrastructure of Cryptosporidium muris (strain RN 66) parasitizing the murine stomach

The ultrastructure of Cryptosporidium muris, which parasitizes the stomach of mice, was studied by transmission electron microscopy. The entire development of the parasite occurred in the microvilli of the surface mucus cells in the gastric glands. The ultrastructural features of the attachment site of C. muris to the host cell differed remarkably from those of C. parvum and its closely related species, which parasitize the intestine of various animals. The size of C. muris was greater at almost every developmental stage than that of C. parvum. These findings confirmed that C. muris and C. parvum are distinct species. The mitochondria, subpellicular microtubules, and Golgi complex were demonstrated in detail. A small invagination in the meront and intravacuolar tubules were found in Cryptosporidium. The wall of each developing oocyst in the parasitophorous vacuole was composed of three layers: the outermost layer was considered to be a true oocyst wall, whereas the middle and innermost layers were assumed to develop into the sporocyst wall. The outermost layer was fragile and disintegrated as the oocyst matured. In excystation in vitro, a suture was seen in a thick layer of the two-layered sporocyst wall of an oocyst (sporocyst wall; see Discussion) that enveloped four sporozoites. The fine structure of the attachment site of the present species to the host cell appears to reveal a unique mode of hostparasite interaction in Cryptosporidium infection.     

Improved excystation protocol for <Emphasis Type="Italic">Eimeria nieschulzi</Emphasis> (Apikomplexa,Coccidia)

Large numbers of sporozoites are a crucial prerequisite for in vitro experiments with Eimeria species. There are no protocols to obtain high amounts of vital purified sporozoites of Eimeria nieschulzi; therefore, an improved excystation protocol is urgently needed. Most excystation procedures for Eimeria oocysts use a mechanical disruption method for the release of sporocysts, assuming that oocyst disruption of Eimeria does not require enzymes (proteases). However, rodent Eimeria oocysts are susceptible to pepsin digestion (Kowalik S, Zahner H (1999) Eimeria separata: method for the excystation of sporozoites. Parasitol Res 85:496–499). Here, we describe a method that combines enzymatic treatment of oocyst walls before the mechanical disruption with glass beads. Using this protocol, we achieved an up to fivefold increase of free viable sporozoites of E. nieschulzi and could significantly shorten the time of excystation. These results confirm the assumption that rodent Eimeria species, in contrast to Eimeria species of birds, possess protease sensitive oocysts.     

Evolution of the endoplasmic reticulum during rat spermiogenesis

The endoplasmic reticulum (ER) was stained selectively by a sequence of uranyl acetate, lead and copper citrate, according to the method of Thiéry and Rambourg ('76), and then investigated in 0.5 to 2.0-μm-thick sections with the transmission electron microscope. Examination of photographic stereopairs allowed a three-dimensional visualization of the ER at various steps of spermiogenesis. During the Golgi and cap phases of spermiogenesis (steps 1–7, classification of Leblond and Clermont, '52a), the ER is distributed throughout the cytoplasm as a three-dimensional network of spherical and tubular cisternae connected by narrow tubules. In addition, a close network of tubular cisternae is located along the convex surface of the Golgi apparatus and lines the plasma membrane. Where the cell membrane joins that of another spermatid to form an intercellular bridge, this network extends across the bridge. During the acrosome phase (steps 8–14), the cytoplasm contains an abundant ER that shows the following modifications: (A) Along the inside and outside of the caudal tube the cisternae form long tubes or plates which run adjacent and parallel to the microtubules. These cisternae are connected by delicate lateral anastomoses; (B) Along the flagellum the ER forms a “fenestrated sleeve” made up of a close network of tubular cisternae; (C) Similar networks are organized as “fenestrated spherules” enclosing large vesicles seen throughout the cytoplasm; (D) At a short distance from the flagellum, the ER cisternae are continuous with a stack of annulate lamellae and an aggregate of radially arranged collapsed cisternae called the “radial body”. During the last or maturation phase (steps 15–19), the ER regresses. Thus, during the final steps in the formation of the flagellum, the ER network fragments and then most of the cisternae disappear from the cytoplasm. The “radial body” is the last element of the ER to be dissolved. Thus the ER undergoes extensive structural modifications during spermiogenesis, suggesting an active contribution of this organelle to the differentiation of the spermatid into a spermatozoon.     

Ultrastructural observations on the sexual stages and oocyst formation inEimeria laureleus (Protozoa,Coccidia) of perch,Perca flavescens,from Lake Sasajewun,Ontario

Ultrastructural features of the sexual stages and oocyst formation ofEimeria laureleus are described from the intestinal epithelium of naturally infected perch (Perca flavescens). Daughter nuclei in maturing microgamonts became aligned in the peripheral cytoplasm opposite thickenings in the limiting membrane. A pair of centrioles, originally arranged at right angles, transformed into basal bodies from which two axonemes arose during the formation of microgametes. Macrogamonts were bound by a trilaminar limiting membrane and were closely invested by the membrane of the parasitophorous vacuole. Maturing macrogamonts had a central nucleus, granular endoplasmic reticulum, amylopectin granules, lipid and peripherally arranged dense, membrane-bound inclusions and mitochondria. Spherical zygotes underwent two divisions to form the sporoblasts, each of which contained, in addition to the above mentioned organelles and inclusions, a large dense refractile body. Each of the four sporocysts contained two residual bodies and closely spaced tubular structures. Slender extensions arose from the sporocyst walls which were about 50 nm thick and were composed of a narrow lucent outer zone and a thicker inner, denser zone which is often striated. The sporocysts were bivalved and joined by a continuous suture. The oocyst boundary was multilaminate and appeared to be composed of components of both the parasite and host cell.