Identification of sporozoite surface proteins and antigens of Eimeria nieschulzi (Apicomplexa)

Sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting, lectin binding, and 125I surface labeling of sporozoites were used to probe sporozoites of the rat coccidian, Eimeria nieschulzi. Analysis of silver stained gels revealed greater than 50 bands. Surface iodination revealed about 14 well labeled, and about 10 weakly labeled but potential, surface proteins. The most heavily labeled surface proteins had molecular masses of 60, 53-54, 45, 28, 23-24, 17, 15, 14, 13, and 12 kD. Following electrophoresis and Western blotting, 2 of the 12 125I labeled lectin probes bound to two bands on the blots, which collectively indicated that two bands were glycosylated. Concanavalin A (ConA) specifically recognized a band at 53 kD, which may represent a surface glycoprotein, and a lectin derived from Osage orange (MPA) bound to a single band at 82-88 kD, that may also be a surface molecule. Immunoblotting using sera collected from rats inoculated orally with oocysts, as well as sera from mice hyperimmunized with sporozoites, revealed that many surface molecules appear to be immunogenic.     

Eimeria melogale n. sp. (Apicomplexa: Eimeriidae) in the Javan ferret-badger (Melogale orientalis)

The Javan ferret-badger Melogale orientalis (Carnivora: Mustelidae: Helictidinae) is a small carnivore endemic to Indonesia. In the family Mustelidae, 10 Eimeria, 12 Cystoisopora, one Isospora, and one Hammondia species are known, but no eimeriid coccidia has been yet described in the subfamily Helictinidae (ferret badgers). Coproscopic examination of Javan ferret-badgers imported into the Czech Republic revealed the presence of coccidian oocysts. Sporulated oocysts differ from other Eimeria known in the family Mustelidae by their small size (12.4–16.1 × 10.4–13.4 μm) and ovoidal shape. Morphological data and phylogenetic analyses of 18S rRNA and COI genes indicated a new species of Eimeria found in faecal samples of Javan ferret badgers. The species is described as E. melogale n. sp.     

Arrested development in Eimeria nieschulzi (Apicomplexa: Eimeriina): results of single infections

Arrested development of Eimeria nieschulzi in the rat was investigated by feeding tissues of infected rats to coccidia-free rats. Intestinal tissue of 33 infected rats was fed on days 9, 10, 11, 14, 17, 21, and 28 PI to 43 uninfected recipients. Eight recipients, which represent 24.2% of the donors, later showed infection. Infections were retained for as long as 21 days after the initial infection. A prepatent period of 6 to 7 days in the recipients allowed the inference that the retained stage was the sporozoite. No infections were observed in 14 recipients fed spleen, lymph nodes, liver, or lung of infected animals.     

Fine Structure of the Oocyst Wall of Eimeria nieschulzi

SYNOPSIS. Oocysts of Eimeria nieschulzi from the laboratory rat, Rattus, norvegicus , were studied by scanning and transmission electron microscopy. Oocysts had a rough outer wall with apparent random depressions. The oocyst wall is composed of 2 layers: an osmiophilic outer layer consisting of a rough external and smooth internal surface, and a relatively thick, electron-lucent inner layer. The outer layer is composed of a dense, coarsely granular matrix. The inner layer consists of homogeneous fine granular material interspersed with coarse osmiophilic granules and contains one closely applied membrane on the outermost surface. Several raised lenticular areas are seen on the coarse outer surface of the inner layer. These layers are 102 (75–128) and 176 (135–204) nm thick, respectively.
The sporocyst wall is thin, consisting of 3 to 4 unit membranes, and measures 27 (18–34) nm thick.     

Suppression of Nippostrongylus brasiliensis (Nematoda)-induced lysophospholipase activity and peripheral eosinophilia by Eimeria nieschulzi (Apicomplexa)

Interspecific interactions between Nippostrongylus brasiliensis and Eimeria nieschulzi were studied by measuring fecal lysophospholipase (LYPH) activity and relative numbers of peripheral eosinophils in rats singly or concurrently infected with one or both parasite species. Three groups of 10 rats each were inoculated with 2 X 10(3) N. brasiliensis L3 larvae and/or 5 X 10(5) E. nieschulzi sporulated oocysts. Groups 1 and 2 were infected with E. nieschulzi or N. brasiliensis, respectively. Group 3 rats were infected first with N. brasiliensis, followed on day 8 postinoculation (PI) with E. nieschulzi. Each rat served as its own control. Results revealed LYPH levels rose steadily in Group 2 rats, reaching significant peaks on days 10 and 12 PI before decreasing to control levels. Lysophospholipase activity in Groups 1 and 3, however, did not differ from control values. Group 2 rats also demonstrated peripheral eosinophilia, with peak values occurring on days 10, 12, 14, and 16 PI, while rats in Groups 1 and 3 exhibited no eosinophilia. These results demonstrate that E. nieschulzi suppressed intestinal LYPH activity and relative peripheral eosinophilia and demonstrate that a host's immune response to a single parasite may be significantly altered when a second parasite species is present.     

Nucleic Acid Precursor Incorporation by Eimeria nieschulzi (Protozoa: Apicomplexa) and Jejunal Villus Epithelium*

Autoradiography was used to investigate incorporation of tritiated adenine, adenosine, guanosine and thymidine by Eimeria nieschulzi and rat jejunal villus epithelial cells. At 2 1/2 days postinoculation, parasitized and control tissues were incubated for 20 min in oxygenated Tyrode's solution (37 C, pH 7.5) containing 30 μCi/ml of each nucleic acid precursor. Treatment of tissues with ribonuclease revealed that E. nieschulzi incorporated label from [³H]adenine primarily into RNA while that from [³H]adenosine and [³H]guanosine was present mainly in DNA. Label from [³H]thymidine was not utilized by parasites. Host villus epithelial cells incorporated label from [³H]purines primarily into RNA. Labeled cytoplasmic RNA was significantly increased in parasitized cells after incubation in [³H]adenine. Tritiated nuclear RNA and cytoplasmic RNA were significantly decreased in parasitized cells after incubation in [³H]adenosine. Incorporation of label from [³H]guanosine was similar for parasitized and control cells. A small quantity of label from each [³H]precursor was incorporated into DNA of villus epithelial cell nuclei.     

Proteins and antigens of merozoites and sporozoites of Eimeria bovis (Apicomplexa)

Proteins and antigens of first-generation merozoites and sporozoites of Eimeria bovis were examined using standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and lactoperoxidase iodination procedures. SDS-PAGE gels revealed both common and unique protein bands in merozoite and sporozoite extracts, ranging in molecular weight (Mr) from 15,000 to 215,000. Nitrocellulose immunoblots of separated proteins, when probed with sera obtained from immunized calves, revealed numerous IgG-binding antigens of Mr 18,000 to 180,000 in merozoites and Mr 28,000 to approximately 118,000 in sporozoites. Although merozoite and sporozoite preparations each contained antigens of different molecular weights, 4 antigens had the same migratory distance in both preparations (Mr 58,000, 70,000, 83,000, 98,000). Of 3 types of immune sera used to probe immunoblots, serum taken from a calf that had been inoculated with oocysts of E. bovis and boosted 10 wk later by subcutaneous injection with 2 X 10(7) live merozoites emulsified in Freund's complete adjuvant consistently identified and reacted more intensely with more antigens of merozoites and sporozoites than the other immune sera tested. Autoradiographic analysis of radioiodinated parasites revealed major surface proteins on merozoites of between 15,000 and 18,000 Mr and 3 surface proteins on sporozoites of Mr 28,000, 77,000, and 183,000. All but the 183,000 protein elicited an IgG antibody response in the host.     

Variation In Surface Proteins In Tetrahymena*

SYNOPSIS. Surface proteins of Tetrahymena were identified by lactoperoxidase iodination, and comparisons were made between a number of strains and species within the genus. an adequate procedure for strain comparisons was found to be solubilization of whole cells following iodination, separation of total cell protein using polyacrylamide gel electrophoresis, and identification of surface proteins by autoradiography of dried gels. the results obtained in the present study show the existence of both interspecific and intraspecific variation in surface proteins of Tetrahymena, but the differences tend to be small within species and large between species. the relation of these cell surface fingerprints to the present taxonomic designations within the genus is discussed. Questions are raised about the functional significance of these surface proteins.     

Suppression of peripheral eosinophilia by the coccidium Eimeria nieschulzi (Apicomplexa: Eimeriidae) in experimentally infected rats

Four groups of 60 rats each were used to examine interspecific interactions between Eimeria nieschulzi and Nippostrongylus brasiliensis. Rats in group 1 served as uninoculated controls. Group 2 rats were each injected subcutaneously with 2.0 X 10(3) L3 larvae of N. brasiliensis. Group 3 rats were each inoculated per os with 2.5 X 10(5) sporulated oocysts of E. nieschulzi. Rats in group 4 were first infected with 2.0 X 10(3) larvae of N. brasiliensis and, at 8 days postinoculation, with 2.5 X 10(5) oocysts of E. nieschulzi. Ten animals from groups 1-3 were sacrificed at 4-day intervals postinoculation and group 4 rats were sacrificed at 4-day intervals beginning after the secondary infection. Blood smears were prepared from each animal to determine differential blood cell counts, bone marrow was examined at the times of peak infection for absolute and relative numbers of eosinophils, portions of the duodenum and jejunum were examined histologically for mast cells, and feces obtained from the cecum and large intestine were examined for ova/gram of feces. Results revealed that relative numbers of peripheral neutrophils and monocytes became elevated during the course of infection for all infected animals, and rats infected with the helminth only also had elevated eosinophil levels. However, rats infected singly with E. nieschulzi, or concurrently with the coccidium and helminth, had peripheral eosinophil levels that were not significantly different from controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Eimeria tenella: Local Antibodies and Interactions with the Sporozoite Surface

.Using fixed sporozoites in a 3-layer immunofluorescence assay (TLIFA), class-specific, parasite-specific antibody responses in chicks to single-pulse infection with Eimeria tenella have been studied in gut contents and bile as well as plasma and feces. After infection with 10³ oocysts, IgA antibody was first detected in the duodenal lumen, then in bile, plasma, cecum, and the distal small intestine. The kinetics of the bile IgA response correlated with that in plasma and peaked 9 days post-infection (d.p.i.); IgM was detected in gut contents and bile as well as plasma, and IgG was occasionally detected in gut contents, especially in the duodenum. In some experiments, IgA was detected in gut contents and bile to at least 21 d.p.i. Infection with 10³ oocysts resulted in an earlier and increased response and relatively high IgG titers in cecal contents. Coproantibody was detected inconsistently and at low titer. When sporozoites that excysted in vitro were incubated in specific, antibody-positive (9 d.p.i.) cecal contents, some complement-mediated IgG-associated anti-sporozoite effects were observed; however, the major effect of cecal contents and the only effect of bile was a non-lethal agglutination of living sporozoites. By fractionation of cecal contents and imtnunoblotting this was confirmed to be IgA mediated; IgA antibodies in cecal contents and bile after infection were shown to bind to sporozoite membrane antigens by surface fluorescence as well as agglutination. Agglutination detected anti-sporozoite antibody in gut contents and bile up to 21 d.p.i., peaking between 7 and 13 d.p.i., corresponding with TLIFA results. The immunofluorescence studies reveal the extremely labile nature of the sporozoite surface antigens and support the hypothesis that naturally elaborated IgA antibodies are involved in the modulation of complexed sporozoite surface antigens.     

Coccidian Parasites (Apicomplexa: Eimeriidae) of Nerodia spp. (Serpentes: Colubridae), with a Description of a New Species of Eimeria

Eimeria conanli n. sp. (Apicomplexa: Eimeriidae) is described from intestinal contents and feces of Nerodia erythrogaster transversa and N harteri harteri from northcentral Texas. Oocysts of the new species are ellipsoid in shape. 17.9 × 13.0(15–21 × 12–15) μm, with a smooth, thin, single-layered wall; shape index 1.4 (1.2–1.5). One to several (usually 2) polar granule(s) and an oocyst residuum are present, but a micropyie is absent. Sporocysts are elongate, 12.9 × 5.2 (13–15 × 5–6) -m, apparently without a true Stieda body structure. Each sporoeyst contains an ellipsoid residuum, 3.9 × 3.2 (3–6 × 2–4) μm, and elongate sporozoites, 11.4 × 2.5 (10–14 × 2–3) μm in situ, each with a spherical or subspherical anterior refractile body and spherical to ellipsoid posterior refractile body. In addition to the new species, oocysts of 4 previously described eimerians from colubrid snakes were found in these hosts.     

Eimeria ornata N. Sp. (Apicomplexa: Eimeriidae) from the Ornate Box Turtle, Terrapene ornata ornata (Reptilia: Testudines), in Texas

Eimeria ornata n. sp. is described from the feces of 6/16 (37.5%) ornate box turtles, Terrapene ornata ornata , in northcentral Texas. Endogenously sporulated oocysts are ellipsoid 17.9 × 15.7(16-21 × 14-18) μm, with a thin, single-layered wall; shape index 1.14 (1.0-1.3). A micropyle is absent but a polar granule was present in one third of the oocysts. An oocyst residuum was present, consisting of numerous small globules situated either in a distinct mass or scattered within the oocyst. The sporocysts are elongate, 11.1 × 5.4 (9-13 × 5-6) μm, with an indistinct Stieda body at 1 pole. A sporocyst residuum is present, consisting either as a compact mass or as scattered granules. The sporozoites are elongate, 9.5 × 2.0 (8-12 × 2) μm, in situ, with spherical anterior and posterior refractile bodies. The new species is distinguished from the similar Eimeria carri Ernst & Forrester, 1973, from eastern box turtles, T. Carolina , by slight differences in oocyst morphology and endogenous sporulation.     

Identification of Surface Antigens of Sarcocystis muris (Coccidia)

Zoites of Sarcocystis muris were recovered from the skeletal muscles of infected mice by trypsin digestion. Extracts of zoites prepared by freeze-thaw, Triton X-100 (0.1%), or a combination of the two treatments contained antigenic components. Testing of these antigens by agar gel diffusion and immunoelectrophoresis against sera from infected mice showed one major precipitin band. SDS-polyacrylamide-gel electrophoresis (SDS-PAGE) of the extracts revealed at least eight detectable polypeptides ranging in molecular weight from 10,000 to 220,000. The antigenic components of the extract were identified by labeling the parasite surface with [¹²⁵I] and precipitation of the [¹²⁵I]-labeled antigens with immune sera. Analysis of the immunoprecipitates by SDS-PAGE and autoradiography revealed three antigens with molecular weights of 27,500, 43,000 and 90,000. The smallest of these was the predominant antigen as suggested by labeling intensity.     

The phylogeny of Goussia and Choleoeimeria (Apicomplexa; Eimeriorina) and the evolution of excystation structures in coccidia

The phylogenetic relationships of Goussia janae and Choleoeimeria sp. were analyzed using the small subunit ribosomal RNA gene (SSU rDNA). This is a first attempt to study the molecular phylogeny of coccidian genera parasitizing strictly poikilotherm hosts. The biliary Eimeria-like coccidia of reptiles classified into the genus Choleoeimeria form a sister clade to the family Eimeriidae, which confirms the separate generic status of the genus Choleoeimeria. The position of Goussia is less robustly resolved, since it forms a trichotomy with the Eimeriidae and Sarcocystidae, or alternatively constitutes the earliest branch of the coccidian lineage. Morphological similarities, namely the extracytoplasmic location of the endogenous stages, and the presence of sutures in the sporocyst wall are discussed in the context of the traditional classification of eimeriids. In contrast to the morphology-based systematics, the monophyly of Goussia and Choleoeimeria is not supported by the SSU rDNA data.     

Isospora Normanlevinei N. Sp. and Isospora Coluzzii N. Sp. (Apicomplexa: Eimeriidae) In Emberiza Cirlus (Cirl Bunting) (Passeriformes-Emberizidae)

ABSTRACT. The morphological characteristics of two new species of Isospora observed in Emberiza cirlus (Cirl Bunting) from Italy are reported. the oocysts of Isospora normanlevinei n. sp. are spherical or sub-spherical, with a smooth double-layered wall, and measure 24.2 times 23.7 (21.0-26.5 times 21.5-25.5) μm; each oocyst contains 2 to 10 polar granules. No micropyle or residuum was observed. the piriform sporocysts measure 19.4 times 11.2 (17.0-21.0 times 10.0-12.5) μm and contain a dispersed residuum. the Stieda body is flat; the substiedal body, with scattered clear and dark granules, may be either symmetrical or asymmetrical. the oocysts of I. coluzzii n. sp. are asymmetrical and rounded shape and measure 28.6 times 24.2 (25.0-31.5 times 21.5-26.0) μm. the oocyst has a double-layered wall and 2 to 3 polar granules. Neither micropyle nor residuum is present. the sub-ellipsoidal sporocyst, measuring 18.2 times 10.0(16.5-20.0 times 9.0-11.0) μm, has a dispersed sporocyst residuum. the Stieda complex is symmetrical.     

Isospora thibetana N. Sp. (Apicomplexa, Eimeriidae), a Parasite of the Tibetan Siskin (Serinus thibetanus=Carduelis thibetanus) (Passeriformes, Fringillidae)

Tibetan siskins are birds native to the Himalayan region often imported into Italy for commercial purposes. Fecal examination of 45 imported subjects with clinical signs of diarrhoea revealed the presence of a large number of coccidian oocysls. After sporulation, accomplished by mixing feces with 2.5 % (w/v) acqueous K₂Cr₂O₇ at room temperature (22° C ± 1° C), exogenous stages of an Isospora species were revealed. The oocysts of this Isospora are spherical, have a bilayered colorless wall, and average 23.24 μm × 23.05 μm; oocyst residuum and micropyle are absent, while an oval polar granule is rarely present. The elliptical sporocysts average 18.44 μm × 10.97 μm and the Stieda body protrudes slightly from the end of the sporocyst. A spherical sporocyst residuum is present though it sometimes consists of scattered granules. The spindle-shaped sporozoites average 11.53 μm × 2.86 μm, and have two refractile bodies. The taxonomic position of the tibetan siskin is controversial. Some authors include this species in the genus Serinus , while others include it in the genus Carduelis. The coccidian species isolated from these tibetan siskins was, for this reason. compared with the Isospora species previously described both in the genus Carduelis and in the genus Serinus. As a result of this comparison a new species. Isospora thibetana , was named. In the intestine of dead subjects, oocysts were found only in the ileum where the mucosa was greatly thickened and presented a heavy leucocytic infiltration consisting mainly of lympho-monocytic cells. A similar infiltration was observed in liver and lungs as well.     

Five new species of Eimeria (Apicomplexa: Eimeriidae) from lizards

Five new species of Eimeria are described from lizards. Eimeria baltrocki n. sp. was found in the berber skink, Eumeces schneideri, from Egypt. The oöcysts are cylindroidal, averaging 38 × 18.3 m, with a single thick oöcyst wall. Most oöcysts possess a single polar granule; a micropyle and oöcyst residuum are absent. The sporocysts are ellipsoidal and average 11.5 × 8.1 m, each with a large, globular sporocyst residuum; the Stieda body is absent. Eimeria anolidis n. sp. is described form the common anole, Anolis carolinensis, from Florida, USA. The oöcysts are cylindroidal and average 31 × 15.8 m with a thick, single-layered oöcyst wall. Two polar granules are usually present; a micropyle and oöcyst residuum absent. The sporocysts are ellipsoidal and average 9.4 × 7.5 m with a globular sporocyst residuum; the Stieda body is absent. Eimeria guyanensis n. sp is recorded in the ameiva, Ameiva ameiva, from Guyana, South America. The oöcysts are spherical to subspherical, average 19.0 × 17.8 m and possess a thick, single-layered oöcyst wall. Numerous polar granules are present (n > 5); a micropyle and oöcyst residuum are absent. The sporocysts are spherical to subspherical, average 7.5 × 7.8 m and possess a compact globular sporocyst residuum; the Stieda body is absent. Eimeria phelsumae n. sp. was recovered from the giant day gecko, Phelsuma madagascariensis grandis, from Madagascar, which harboured a simultaneous infection of E. brygooi. The oöcysts measured 32 × 15 m and are cylindroidal without polar granules, a micropyle or oöcyst residuum, or a Steida body. The sporocysts are ellipsoidal and average 9.8 × 7 m, with a loosely clumped, granular sporocyst residuum; the Steida body is absent. Eimeria leiocephali n. sp. was discovered in the faeces of the ornate ground iguana, Leiocephalus barahonensis, from Haiti. The oöcysts are spherical to subspherical, 21 × 19 m, and contain a number of polar granules (n > 5); a micropyle and oöcyst residuum are absent. The sporocysts are spherical, 8 m in diameter and lack a sporocyst residuum. Eimeria turcicus and E. lineri were found in faeces of Hemidactylus turcicus turcicus from the host's country of origin, Turkey.     

Both CP15 and CP25 are left as trails behind gliding sporozoites of Cryptosporidium parvum (Apicomplexa)

Abstract Sporozoites of Cryptosporidium parvum were examined after gliding upon glass microscope slides using monoclonal antibodies to the 15 and 25 kDa surface molecules and immunogold-silver enhancement. Both antibodies bound to surface antigen deposited as trails behind parasites, suggesting that both surface molecules are involved in substrate attachment.