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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.

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Infection of mice with Neospora caninum (Protozoa: Apicomplexa) does not protect against challenge with Toxoplasma gondii.

Neospora caninum and Toxoplasma gondii are structurally related protozoal parasites of mammals that may cause abortion and neonatal morbidity and mortality. Groups of mice were subcutaneously inoculated with 10(5) live zoites of the NC-1 or NC-3 isolates of N. caninum and reinoculated with an identical number of live zoites 2 weeks later. Groups of mice which were injected subcutaneously with Hanks balanced salt solution served as controls. Three weeks after the final N. caninum inoculation, one-half of the mice were inoculated subcutaneously with 2.5 x 10(4) zoites of the RH isolate of T. gondii and the other half were inoculated subcutaneously with 2.5 x 10(4) zoites of the GT-1 isolate of T. gondii. Serum samples taken from mice on the day of T. gondii inoculation were negative for specific antibodies to T. gondii, but mice inoculated with N. caninum had reciprocal titers of greater than or equal to 800 to this protozoan. All of the mice died after challenge with T. gondii, and no significant differences (P greater than 0.05) between the survival times of mice inoculated with either isolate of N. caninum and those of control mice were seen. This study indicates that N. caninum and T. gondii are distinct biologic entities and not closely related isolates.

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Cryptosporidium parvum (Apicomplexa: Cryptosporidiidae) oocyst and sporozoite antigens recognized by bovine colostral antibodies.

Colostral whey from seven hyperimmunized and two control cows (hyperimmune bovine colostrum) was examined by Western immunoblotting for the presence of antibody against oocysts and sporozoites of Cryptosporidium parvum, using rabbit anti-bovine immunoglobulin A (IgA), IgG1, IgG2, and IgM antibodies, followed by a horseradish peroxidase goat anti-rabbit polyvalent antibody. Although considerable variation was found in binding activity between cows on different immunization protocols, IgA and IgG1 in whey recognized a greater variety of C. parvum antigens than did IgG2 and IgM. A band at 9 to 10 kilodaltons appeared unique in that it was recognized only by IgA.

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Characterization of microneme antigens of Cryptosporidium parvum (Protozoa, Apicomplexa).

Two monoclonal antibodies (MAbs) raised against purified excysted oocysts and sporozoites of cryptosporidium parvum reacted in an immunofluorescence assay with antigens located at the anterior pole of the zoites. On Western blots of purified oocysts, these MAbs reacted with a series of bands between 210 and 40 kDa; several of these bands were recognized by both MAbs; others were specific. One MAb (TOU) did not react after periodic acid treatment and was therefore considered to recognize a carbohydrate epitope; as determined by immunoelectron microscopy, this MAb reacted on micronemes of sporozoites and merozoites and also with the peripheral cytoplasm and the parasitophorous vacuole of trophozoites and macrogametes. The other MAb (HAD) reacted with an epitope that was insensitive to periodate treatment but did not react in the immunoelectron microscopy assay. However, the similar labeling pattern obtained with the immunofluorescence assay with both MAbs and the fact that the two antibodies share common bands on Western immunoblots suggest that both MAbs react with molecules located in Cryptosporidium micronemes, one reacting with a glycannic epitope and the other reacting with a peptidic epitope.

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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.

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How individual cells develop from a syncytium: merogony in Theileria parva (Apicomplexa).

The central problem for Theileria parva during merogony is how to form numerous individual, uninucleate merozoites from a syncytial schizont so that each merozoite contains a single nucleus and a prescribed assortment of organelles. The way T. parva packages all the requisite organelles into free merozoites is by binding these organelles to the nuclear envelope, which in turn becomes associated, both directly and through the rhoptry complex, with the schizont plasma membrane. Formation of the merozoites occurs in a synchronous manner by a budding process. The merozoites develop with the rhoptry complex at the apical end by the progressive, outward evagination of the schizont plasma membrane. This evagination of the plasma membrane is associated with, and presumably induced by, the development of an orderly array of tubules that originate from the apical end and progressively form a longitudinal basket enclosing first the rhoptry complex, then the mitochondria and ribosomes, and finally the nucleus. The process of merogony is compared to sporogony within the tick salivary gland and with the differentiation of the intra-erythrocytic piroplasm stage. Because all three processes occur by a morphologically similar mechanism, the possibility that the parasite uses a single cassette of genes to perform each of these similar processes is discussed.

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Ultrastructural study of meronts and gamonts of Choleoeimeria rochalimai (Apicomplexa: Eimeriidae) developing in the gall bladder of the gecko Hemidactylus mabouia from Brazil.

Endogenous development of Choleoeimeria rochalimai (Carini et Pinto, 1926) Lainson et Paperna, 1999 in the gall bladder of Hemidactylus mabouia (Moreau de Jonnes, 1818) from Belém, Brazil is reported at the fine structural level. Meronts and gamonts develop in the epithelial cells of the gall bladder. Infected cells become enlarged and displaced above the epithelial layer. Developing merozoites, dividing meronts and succession of developing microgamonts from initial nuclear division up to final microgamete differentiation are described. In addition towall forming bodies, mature macrogamonts possess a large inclusion or cisterna with fine granular contents.

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Eimeria burdai sp. n. (Apicomplexa: Eimeriidae), a new parasite species from subterranean African silvery mole-rat, Heliophobius argenteocinereus.

A new coccidian parasite of the genus Eimeria Schneider, 1875 is described from the subterranean African silvery mole-rat Heliophobius argenteocinereus Peters, 1846. Oocysts of Eimeria burdai sp. n. were subspherical to broadly ellipsoidal 17.8 (16-19) x 14.1 (12-15), with a shape index 1.2 (1.1-1.4). Oocyst wall was bilayered, smooth and colourless, approximately 1.0 thick. Outer layer was significantly thicker than inner one. A micropyle and oocyst residuum were absent. One or two ellipsoidal or spherical polar granules were present. Sporocysts were ellipsoidal, 10.8 (9-12) x 6.2 (5-8) with a shape index 1.7 (1.5-1.9). Sporocyst wall was single-layered, thin, smooth and colourless, with small Stieda body at the pointed end. In freshly sporulated oocysts, spherical sporocyst residuum was composed of small granules enclosed by a thin membrane. Sporozoites were elongate, lying length-wise in the long axis of the sporocyst, partially curled around each other, with single large refractile body located posteriorly. Faintly distinguishable nucleus was in the central part of the sporozoite. This eimerian represents the first coccidian species described from subterranean African silvery mole-rat (Rodentia: Bathyergidae).

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A description of two new species of coccidia (Apicomplexa: Eimeriidae) from African reptiles with nomenclatural corrections for two Caryospora and one Eimeria species from snakes.

Two new species of coccidian parasites are described from African reptiles. Oocysts of Eimeria foulshami sp. n. from the plated lizard Gerrhosaurus major bottegoi Del Prato of Sudan are ellipsoidal, 24.1 x 14.9 (23-26.5 x 14-17.8) microm with a bilayered, colourless oocyst wall and lack polar granules. The ellipsoidal sporocysts average 8.6 x 4.6 (7-10.6 x 4.4-7) microm and possess a prominent, globular, sporocyst residuum. Oocysts of Caryospora regentensis sp. n. from the Eastern green mamba Dendroaspis augusticeps Smith, 1849 [corrected] of Kenya are spherical to subspherical, 16.8 x 16.4 (16-17.6 x 15-17.2) microm with a bilayered oocyst wall and a single polar granule. The ellipsoidal sporocysts average 13.0 x 10.3 (10.2-14 x 9.2-11) microm and possess a Stieda and substieda body and a prominent globular sporocyst residuum. Oocysts of Caryospora legeri Hoare, 1933 are reported from a hissing sand snake, Psammophis sibilans sibilans L. from Nigeria, representing a new geographical record. The oocysts are slightly larger than the type, but otherwise identical. Caryospora psammophi Bray, 1960 and C. hermae Bray, 1960 from Psammophis sibilans phillipsi, oocysts of which are morphologically similar to and overlap in dimensions with C. legeri Hoare, 1933, are synonymised with the latter species. Eimeria samiae Iskander et Tadros, 1979 is emended to E. samyadeli to reflect the gender of the person the species was named after and because E. sani is preoccupied. In addition to these findings, Eimeria bohemi Modrý, Slapeta et Koudela, 2000 and oocysts of an unidentified spherical Eimieria sp. are reported from Chamaeleo dilepis dilepis Leach from Cameroon.

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Caryospora varaniornati sp. n. (Apicomplexa: Eimeriidae) in the Nile monitor, Varanus (Polydaedalus) niloticus species complex.

Parasitological examination of two ornate Nile monitors Varanus ornatus (Daudin, 1803) imported from Benin revealed the presence of a new species of Caryospora. Oocysts of Caryospora varaniornati sp. n. are spherical to slightly subspherical, 12.0 (11-12.5) x 11.5 (11-12) microm, without amicropyle and oocyst residuum, and occasionally possessing one small polar granule. Sporocysts are broadly ellipsoidal, 8.8 (8.5-9.5) x 6.7 (6.5-7) microm; a lentil-like Stieda body is present, ca. 0.5 x 1 microm; substieda body not visible. Experimental infection of a closely related host, Varanus niloticus (L.), did not lead to the oocyst excretion despite the fact that one of the experimentally inoculated monitors was immunosuppressed by dexamethasone. Histological examination did not reveal stages of coccidian development. Therefore, it is possible that C. varaniornati is strictly host specific.

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Experimental transmission of Caryospora kutzeri (Apicomplexa: Eimeriidae) by rodent hosts.

Four laboratory-hatched European kestrels Falco tinnunculus L. were fed on laboratory mice and common voles Microtus arvalis Pallas previously inoculated with different doses of sporulated oocysts of Caryospora kutzeri Böer, 1982. Two kestrels that were fed infected mice shed C. kurtzeri oocysts 6 days after ingesting murine tissues. To compare direct and indirect transmissions, two of the kestrels were subsequently directly inoculated with 10(5) sporulated C. kutzeri oocysts and became patent on days 8 and 9 and shed caryosporan oocysts up to day 25 post inoculation. Additionally, four mice were inoculated with 10(6) oocysts in order to examine mouse tissues for the presence of developmental stages of C. kutzeri. No coccidian stages were found in the tissues of inoculated mice. The experiment showed that developmental stages of C. kutzeri are able to survive in mouse tissues and cause infection of suitable host after their ingestion.

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Description of Eimeria arabukosokokensis sp. n. (Apicomplexa: eimeriidae) from Telescopus semiannulatus (Serpentes: Colubridae) with notes on eimerian coccidia from snakes of Eastern Kenya.

Parasitological examination of faeces of 26 snakes kept in Bio-Ken Snake Farm, Watamu, Kenya revealed new species of Eimeria Schneider, 1875 in Telescopus semiannulatus Smith, 1849. Oocysts of Eimeria arabukosokokensis sp. n. are cylindrical 26.8 (25-29) x 15.1 (14-16) microm with smooth, bilayered oocyst wall and a single polar granule. The broadly ellipsoidal sporocysts average 9.3 (8.5-10) x 7.1 (6.5-7.5) microm and possess single-layered wall composed of two plates joined by longitudinal suture. Caryospora cf. regentensis Daszak et Ball, 2001 is reported from Dendroaspis angusticeps (Smith, 1849) and two additional forms of Caryospora Léger, 1904 are reported and morphologically characterised from a single specimen of Psammophis orientalis Broadley, 1977. Systematic status of Caryospora spp. in sub-Saharan Psammophis Boie, 1827 is discusses and all species reported by various authors to date are suggested to be treated as species inquirendae until more detailed data on these parasites and their hosts are available.

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The endogenous development, described by light and electron microscopy, of Eimeria jamescooki sp. n. (Apicomplexa: Eimeriidae) from the skink Cryptoblepharus virgatus.

Eimeria jamescooki sp. n. was recovered from the skink Cryptoblepharus virgatus (Garman) found on the grounds of James Cook University, Townsville (type locality), North Queensland, Australia. Oocysts were 17.5-25.0 (22.1 +/- 1.9) x 15-22.5 (17.7 +/- 1.6) microm and sporocysts 6.25-10.0 (7.9 +/- 1.15) x 3.75-6.25 (5.3 +/- 1.0) microm in size. Endogenous stages are described from histological material examined by light microscope and by transmission electron microscope. Both merogony stages and gamonts were found to develop in the cytoplasm of the anterior gut mucosal epithelium. Meront progeny were comprised of 10 to 21 merozoites. Premature macrogamonts were elongate; some host cells contained two elongate macrogamonts. Unique to the presently described species were the Golgi "plaques" and an enclosure of tubuli. Mature macrogamonts and young oocysts ranged in size from 14 x 7 to 21 x 11 microm and contained two types of wall-forming bodies, canaliculi and amylopectin granules. Differentiating microgamonts conformed in fine structure with that observed in other eimerians. Their sizes increased from 15.4 x 4.2 to 28 x 8.4 microm while dividing to over 70 nuclei, which formed a corresponding yield of microgametes.

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Rheumatoid factor-like IgM in Plasmodium berghei (Apicomplexa: Haemosporida) infections of BALB/c mice.

Groups of female BALB/c mice infected by intravenous injection with 50 erythrocytes containing Plasmodium berghei Vincke et Lips, 1948 were sacrificed on days 3 through 12 after infection. Rheumatoid factor-like IgM (RF-IgM) and parasite-specific IgG levels were determined by enzyme-linked immunosorbent assay in serum specimens and in culture medium removed from spleen cell cultures established at sacrifice. All four mouse IgG subisotypes were recognized by RF-IgM molecules induced by Plasmodium berghei infection, and in this regard, the parasite-induced RF-IgM response resembled that induced by lipopolysaccharide polyclonal activation. Plasmodium berghei infection resulted in a biphasic RF-IgM response, with infected animals demonstrating significantly increased levels of RF-IgM early in the infection and significantly decreased levels late in the infection, compared to uninfected control mice. The decreased levels of RF-IgM observed late in infection correlated with increasing parasitaemia levels, and were primarily due to a decrease in RF-IgM specific for mouse IgG2a. Late infection levels of RF-IgM specific for IgGI, IgG2b, and IgG3 were not significantly different from those of control animals.

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Haemogregarina bigemina (Protozoa: Apicomplexa: Adeleorina)--past, present and future.

This paper reviews past, current and likely future research on the fish haemogregarine, Haemogregarina bigemina Laveran et Mesnil, 1901. Recorded from 96 species of fishes, across 70 genera and 34 families, this broad distribution for H. bigemina is questioned. In its type hosts and other fishes, the parasite undergoes intraerythrocytic binary fission, finally forming mature paired gamonts. An intraleukocytic phase is also reported, but not from the type hosts. This paper asks whether stages from the white cell series are truly H. bigemina. A future aim should be to compare the molecular constitution of so-called H. bigemina from a number of locations to determine whether all represent the same species. The transmission of H. bigemina between fishes is also considered. Past studies show that young fish acquire the haemogregarine when close to metamorphosis, but vertical and faecal-oral transmission seem unlikely. Some fish haemogregarines are leech-transmitted, but where fish populations with H. bigemina have been studied, these annelids are largely absent. However, haematophagous larval gnathiid isopods occur on such fishes and may be readily eaten by them. Sequential squashes of gnathiids from fishes with H. bigemina have demonstrated development of the haemogregarine in these isopods. Examination of histological sections through gnathiids is now underway to determine the precise development sites of the haemogregarine, particularly whether merozoites finally invade the salivary glands. To assist in this procedure and to clarify the internal anatomy of gnathiids, 3D visualisation of stacked, serial histological sections is being undertaken. Biological transmission experiments should follow these processes.

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Mice serve as paratenic hosts for the transmission of Caryospora duszynskii (Apicomplexa: Eimeriidae) between snakes of the genus Elaphe.

Caryospora duszynskii Upton, Current et Barnard, 1984 was successfully transmitted to snakes of the genus Elaphe by feeding them previously infected mice. Fifty thousand oocysts were orally administered to two mouse strains, BALB/c and Crl:CD-1(ICR)BR, which were subsequently fed to captive-born coccidia-free Elaphe guttata (L.) in two respective independent experiments. Both E. guttata expelled C. duszynskii oocysts in their faeces, beginning on day 18 and 26 post infection (p.i.) and shed oocysts continuously through the end of the experiment, day 230 and 135 p.i., respectively. There were no parasitic stages or lesions in mice, as revealed by histological examination. Experiments proved that rodents serve as paratenic hosts for C. duszynskii. In summary we discuss the life-cycle strategies of Caryospora spp. in reptiles and present three general modes of their development.

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Observations on some avian Coccidia (Apicomplexa: Eimeriidae) in Amazonian Brazil.

Oocysts of Eimeria porphyrulae n.sp. are described in faeces of Porphyrula martinica (Aves: Gruiformes: Rallidae). They are ellipsoidal to oval, 22.4 x 17.7 (20.0-23.7 x 16.2-18.7) microns, shape-index (length/width) 1.3. Oocyst wall about 1.25 microns thick, colourless, with two layers: inner one prominently striated. Micropyle and sub-micropylar granule present: no oocyst residuum. Sporocysts 17.5 x 9.0 (17.0-19.0 x 8.0-10.0) microns, shape-index 1.9, with inconspicuous Stieda/sub-Stieda bodies. Sporocyst residuum of scattered granules, sometimes a compact mass: sporozoites with two refractile bodies. Eimeria crypturelli n.sp. is described in faeces of Crypturellus soui (Tinamiformes: Tinamidae). Oocysts ellipsoidal-oval, 20.75 x 14.5 (17.5-25.0 x 11.25-21.25) microns, shape-index 1.4. Oocyst wall about 1.25 microns thick and bi-layered: inner layer faintly striated. Micropyle present, with oocyst residuum immediately below: single polar body rarely present. Sporocysts 13.0 x 7.5 (12.5-13.75 x 7.5-8.1) microns, shape-index 1.7, with a Stieda body but seemingly no sub-Stieda. Sporocyst residuum compact: sporozoites with two refractile bodies. Isospora cacici n.sp. is recorded from faeces of Cacicus cela cela (Passeriformes: Icteridae). Oocysts subspherical-spherical, 26.5 x 23.7 (22.5-27.5 x 20.0-26.2) microns, shape-index 1.1. Wall a single, colourless layer about 1.5 microns thick. No micropyle or oocyst residuum: 1-2 polar bodies. Sporocysts ellipsoidal, 17.7 x 12.5 (17.5-18.75 x 11.25-13.75) microns, shape-index 1.4, with pronounced Stieda/sub-Stieda bodies: residuum compact and sporozoites with two refractile bodies. Isospora thraupis n.sp. is described from faeces of Thraupis palmarum melanoptera (Passeriformes: Thraupidae).(ABSTRACT TRUNCATED AT 250 WORDS)

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