Toxoplasma gondii: Microassay to differentiate toxoplasma inhibiting factor and interleukin 2

Using a sensitive, economical, and reproducible microassay, the relationship of toxoplasma inhibiting factor to interleukin 2 has been examined. The assay developed took advantage of the observation that (1) Toxoplasma gondii tachyzoites replicated efficiently in the murine monocytic cell line, RAW 264; (2) treatment of RAW 264 cells with toxoplasma inhibiting factor prevented intracellular replication of the parasite to an extent similar to that observed with identical treatment of freshly isolated murine peritoneal exudate cells; and (3) [³H]uracil incorporation was an efficacious means to quantify replication (or inhibition of replication) of tachyzoites within the cell line. Although toxoplasma inhibiting factor and interleukin 2 were both present in the same lectin -and antigen-stimulated splenocyte supernatant fluids, results from microassays strongly suggested that the molecules were two distinct entities.     

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.     

肠艾美耳球虫(Eimeria intestinalis)内生发育阶段超微结构研究:Ⅱ.大配子的发育及卵囊壁的形成

肠艾美耳球虫(Eimeria intestinalis)大配子体(Macrogametocyte)带虫空泡内有许多泡内小管。大配子体外被单层限制膜,核大,有一明显的核仁。两种成囊颗粒几乎同时出现。发育中的大配子体胞质中出现多糖和脂肪小体,并逐渐增多。大配子表面被有两层膜。卵囊壁形成的标志是大配子表面的两层膜松弛地向带虫空泡内推移,成囊颗粒Ⅰ形成卵囊壁的外层,成囊颗粒Ⅰ的物质形成卵囊壁的内层,在内层之下又有一颗粒层的形成和消失。     

In vitro cultivation methods for coccidian parasite research

The subclass Coccidia comprises a large group of protozoan parasites, including important pathogens of humans and animals such as Toxoplasma gondii, Neospora caninum, Eimeria spp., and Cystoisospora spp. Their life cycle includes a switch from asexual to sexual stages and is often restricted to a single host species. Current research on coccidian parasites focuses on cell biology and the underlying mechanisms of protein expression and trafficking in different life stages, host cell invasion and host-parasite interactions. Furthermore, novel anticoccidial drug targets are evaluated. Given the variety of research questions and the requirement to reduce and replace animal experimentation, in vitro cultivation of Coccidia needs to be further developed and refined to meet these requirements. For these purposes, established culture systems are constantly improved. In addition, new in vitro culture systems lately gained considerable importance in research on Coccidia. Well established and optimized in vitro cultures of monolayer cells can support the viability and development of parasite stages and even allow completion of the life cycle in vitro, as shown for Cystoisospora suis and Eimeria tenella. Furthermore, new three-dimensional cell culture models are used for propagation of Cryptosporidium spp. (close relatives of the coccidians), and the infection of three-dimensional organoids with T. gondii also gained popularity as the interaction between the parasite and host tissue can be studied in more detail. The latest advances in three-dimensional culture systems are organ-on-a-chip models, that to date have only been tested for T. gondii but promise to accelerate research in other coccidians. Lastly, the completion of the life cycle of C. suis and Cryptosporidium parvum was reported to continue in a host cell-free environment following the first occurrence of asexual stages. Such axenic cultures are becoming increasingly available and open new avenues for research on parasite life cycle stages and novel intervention strategies.     

Dynamics of cytokine production during coccidial infections in chickens: colony-stimulating factors and interferon

Abstract We assayed two classes of immunoregulatory cytokines, colony-stimulating factors (CSF) and interferon (IFN), during and immediately after a primary coccidial infection in chickens. Coccidial infection induces significant alterations in serum colony-stimulating activity (CSA) and these alterations immediately precede the characteristic biphasic leukocytosis. CSA rose sharply during the first 24 h post-inoculation (PI), but returned to control levels by 48 h PL. At this time, we detected an increase in peripheral blood leukocytes which peaked at 96 h PI. A second phase of CSA increase began 96 h PI and peaked at 120–144 h PI which again preceded the second phase of leukocytosis. We also examined the production of IFN during the first 20 days PI. Splenic T cells from Eimeria maxima -infected chickens produced significantly less IFN on day 5 PI compared to T cells from the coccidia-free controls. By days 10 and 15 PI, there was no significant difference in IFN production between the T cells of infected and non-infected chickens. However, by day 20 PI, IFN production by the T cells of the infected birds produced significantly more IFN than the control T cells. The results of our studies indicated the differential production of two different cytokines by chickens during and following a primary coccidial infection. Based on these experiments, CSF may be some of the first cytokines produced during an E. maxima -infection, while IFN may be one of the later cytokines produced.     

The methylerythritol phosphate pathway for isoprenoid biosynthesis in coccidia: presence and sensitivity to fosmidomycin

The apicoplast is a recently discovered, plastid-like organelle present in most apicomplexa. The methylerythritol phosphate (MEP) pathway involved in isoprenoid biosynthesis is one of the metabolic pathways associated with the apicoplast, and is a new promising therapeutic target in Plasmodium falciparum. Here, we check the presence of isoprenoid genes in four coccidian parasites according to genome database searches. Cryptosporidium parvum and C. hominis, which have no plastid genome, lack the MEP pathway. In contrast, gene expression studies suggest that this metabolic pathway is present in several development stages of Eimeria tenella and in tachyzoites of Toxoplasma gondii. We studied the potential of fosmidomycin, an antimalarial drug blocking the MEP pathway, to inhibit E. tenella and T. gondii growth in vitro. The drug was poorly effective even at high concentrations. Thus, both fosmidomycin sensitivity and isoprenoid metabolism differs substantially between apicomplexan species.     

Chasing coccidia--new tools enter the race

The 8th International Coccidiosis Conference, held on 9--13 July 2001 in Palm Cove, Australia, was a showcase of the latest studies on widely known coccidia, including Eimeria and Toxoplasma in addition to the emerging or re-emerging parasites such as Neospora, Cryptosporidium and Cyclospora. This meeting was staged in conjunction with the Annual Scientific Meeting of the Australian Society for Parasitology.     

Recent advances in the search for new anti-coccidial drugs

Coccidia provide a rich hunting ground for drug-designers, as there are significant biochemical differences between the parasites and their hosts. Recent years have brought the discovery of the plastid and its possible metabolic machinery, characterisation of acidocalcisomes, reports on the apparent absence from some coccidia of a typical mitochondrion, and the discovery of the mannitol cycle and shikimate pathway in the parasites. Moreover, modern technologies such as genomics and proteomics are bringing new insights into the biochemistry of coccidia and highlighting possible drug targets in abundance. A major issue for would-be drug discoverers is to decide upon the targets to prioritise. This review provides an update on recent findings on how coccidia differ biochemically from vertebrates. It includes discoveries within coccidian parasites themselves but also uses findings in Plasmodium to provide an overview of biochemical features that may be characteristics of many apicomplexan parasites and so potential targets for broad-spectrum drugs.     

Extraintestinal gamogony of Aggregata octopiana in the reared common octopus (Octopus vulgaris) (Cephalopoda: Octopodidae)

Aggregata octopiana (Apicomplexa, Aggregatidae) is the most prevalent coccidian in the wild common octopus (Octopus vulgaris), whose heteroxenous life cycle includes gamogony and sporogony undergoing in the octopus digestive tract. In the infected reared octopi, we observed an unusual extraintestinal distribution of the coccidian, with both gamogony and sporogony ongoing in dermal and gill tissue. Oocysts and macrogamonts were embedded in the dermal connective tissue of octopian arms, demarcated by a thin cyst wall or multilayered dark membrane. In gill connective and epithelial tissue all developmental stages were observed, eliciting hemocytic infiltration. Sometimes a complete substitution of the tissue by cysts and developmental stages occurred, resulting in necrosis of gill tissue.     

Prevalence and intensity of excretion of coccidial oocysts in migrating passerines on Helgoland

Prevalence and intensity of excretion of coccidial oocysts in actively migrating passerines (Turdus philomelos, Fringilla coelebs, Sylvia borin, Phoenicurus phoenicurus) were assessed in spring and autumn 2001 during stopovers on Helgoland. All examined species showed low prevalences of oocysts in spring (28–33%) and high prevalences in autumn migration (66–92%). In autumn, there are significant species-specific differences in prevalence and, partially, in intensity of excretion. Ground-feeding Chaffinches most commonly and most intensively shed oocysts (maximal 2,7×10⁶ oocysts per g fresh faeces) indicating that feeding style may influence the parasite load. No significant correlation between parasite load, age, and body condition (fat depots, breast muscle shape, body mass) was found. The connection between feeding style, age, migration, immunocompetence, and survival of passerine hosts on one hand and the particularities of coccidial pathogenicity on the other are discussed.