Multimodal analysis of Plasmodium knowlesi-infected erythrocytes reveals large invaginations, swelling of the host cell, and rheological defects.

The simian parasite Plasmodium knowlesi causes severe and fatal malaria infections in humans, but the process of host cell remodelling that underpins the pathology of this zoonotic parasite is only poorly understood. We have used serial block-face scanning electron microscopy to explore the topography of P. knowlesi-infected red blood cells (RBCs) at different stages of asexual development. The parasite elaborates large flattened cisternae (Sinton Mulligan's clefts) and tubular vesicles in the host cell cytoplasm, as well as parasitophorous vacuole membrane bulges and blebs, and caveolar structures at the RBC membrane. Large invaginations of host RBC cytoplasm are formed early in development, both from classical cytostomal structures and from larger stabilised pores. Although degradation of haemoglobin is observed in multiple disconnected digestive vacuoles, the persistence of large invaginations during development suggests inefficient consumption of the host cell cytoplasm. The parasite eventually occupies ~40% of the host RBC volume, inducing a 20% increase in volume of the host RBC and an 11% decrease in the surface area to volume ratio, which collectively decreases the ability of the P. knowlesi-infected RBCs to enter small capillaries of a human erythrocyte microchannel analyser. Ektacytometry reveals a markedly decreased deformability, whereas correlative light microscopy/scanning electron microscopy and python-based skeleton analysis (Skan) reveal modifications to the surface of infected RBCs that underpin these physical changes. We show that P. knowlesi-infected RBCs are refractory to treatment with sorbitol lysis but are hypersensitive to hypotonic lysis. The observed physical changes in the host RBCs may underpin the pathology observed in patients infected with P. knowlesi.

Isolation, molecular characterization, and in vitro schizogonic development of Sarcocystis sp. ex Accipiter cooperii from a naturally infected Cooper's hawk (Accipiter cooperii).

Raptors serve as the definitive host for several Sarcocystis species. The complete life cycles of only a few of these Sarcocystis species that use birds of prey as definitive hosts have been described. In the present study, Sarcocystis species sporocysts were obtained from the intestine of a Cooper's hawk (Accipiter cooperii) and were used to infect cell cultures of African green monkey kidney cells to isolate a continuous culture and describe asexual stages of the parasite. Two clones of the parasite were obtained by limiting dilution. Asexual stages were used to obtain DNA for molecular classification and identification. PCR amplification and sequencing were done at three nuclear ribosomal DNA loci; 18S rRNA, 28S rRNA, and ITS-1, and the mitochondrial cytochrome c oxidase subunit 1 (cox1) locus. Examination of clonal isolates of the parasite indicated a single species related to S. columbae (termed Sarcocystis sp. ex Accipiter cooperii) was present in the Cooper's hawk. Our results document for the first time Sarcocystis sp. ex A. cooperii occurs naturally in an unknown intermediate host in North America and that Cooper's hawks (A. cooperii) are a natural definitive host.

Pellicle formation in the malaria parasite.

The intraerythrocytic developmental cycle of Plasmodium falciparum is completed with the release of up to 32 invasive daughter cells, the merozoites, into the blood stream. Before release, the final step of merozoite development is the assembly of the cortical pellicle, a multi-layered membrane structure. This unique apicomplexan feature includes the inner membrane complex (IMC) and the parasite's plasma membrane. A dynamic ring structure, referred to as the basal complex, is part of the IMC and helps to divide organelles and abscises in the maturing daughter cells. Here, we analyze the dynamics of the basal complex of P. falciparum. We report on a novel transmembrane protein of the basal complex termed BTP1, which is specific to the genus Plasmodium. It colocalizes with the known basal complex marker protein MORN1 and shows distinct dynamics as well as localization when compared to other IMC proteins during schizogony. Using a parasite plasma membrane marker cell line, we correlate dynamics of the basal complex with the acquisition of the maternal membrane. We show that plasma membrane invagination and IMC propagation are interlinked during the final steps of cell division.

Ultrastructural effects of acetamizuril on endogenous phases of Eimeria tenella.

To explore the primary stage or site of action of acetamizuril (AZL), a novel triazine anticoccidial compound, the ultrastructural development of Eimeria tenella at different endogenous stages was studied in experimentally infected chickens treated with a single oral dose of 15 mg/kg AZL. As a result of drug action, the differentiations of second-generation schizonts and microgamonts were largely inhibited and merozoites became irregular in shape. Meanwhile, the outer membrane blistering and perinuclear space enlargement were obvious in the second-generation schizonts and microgamonts, which were never observed in the classic triazine anticoccidiosis drug diclazuril-treated E. tenella. The chromatin aggregation, anachromasis, and marginalization were visible in merozoites and microgamonts. Furthermore, the abnormal evagination of microgametes finally resulted in the degeneration of microgamonts and the failure of subsequent fertilization. The most marked micromorphological alteration occurring in the macrogamonts was the WFB2. Even if the fertilization occurred, the formation of oocyst wall became malformed and the zygote proceeded to the obvious degeneration. In addition, mitochondria swelling and cytoplasm vacuolization were discerned in respective intracellular stages, while endoplasmic reticulum and Golgi body swelling was less seen. These alterations may be the causes leading to the final death of E. tenella and also provide some information for further exploring the mechanism of action of AZL at the molecular level.

Description of a New Haemogregarine, Haemogregarina sundarbanensis n. sp. (Apicomplexa: Haemogregarinidae) from Mud Turtle of Sundarban Regions, West Bengal, India.

OBJECTIVE: The aim of this study was to provide a description of a new haemogregarine, Haemogregarina sundarbanensis n. sp., from mud turtles collected from Sundarban regions, West Bengal, India. METHODS: The turtles were collected from fish markets near Canning, South 24-Parganas District, West Bengal. Thin blood films were prepared using the blood drawn from the head and feet. The blood films were then air-dried, fixed in absolute methanol, and stained with Giemsa. RESULTS: The erythrocytic stages could be differentiated into small and large forms. The young gamonts measured 7.896×3.469 µm, microgamonts measured 8.876×4.425 µm, and macrogamonts measured 8.919×4.272 µm. The cytoplasm of the macrogamonts was stained deep blue with Giemsa, and many metachromatic granules are found uniformly distributed. The oval or rounded nucleus was central in position and stained deep red with Giemsa stain, whereas in microgamonts, the nucleus was not at all compact but with a few dispersed chromatin granules. CONCLUSION: The prevalence of infection was found to be 33.3% (5/15). The infected erythrocytes reveal shape alteration, marginal and atrophic nucleus, and were larger than non-parasitized erythrocytes. The cytoplasm of the infected erythrocytes stained darker than that of the uninfected erythrocytes.

In vitro cultivation of Hematodinium sp. isolated from Atlantic snow crab, Chionoecetes opilio: partial characterization of late developmental stages.

Hematodinium is a parasitic dinoflagellate of numerous crustacean species, including the economically important Atlantic snow crab, Chionoecetes opilio. The parasite was cultured in vitro in modified Nephrops medium at 0 °C and a partial characterization of the life stages was accomplished using light and transmission electron microscopy (TEM). In haemolymph from heavily infected snow crabs two life stages were detected; amoeboid trophonts and sporonts. During in vitro cultivation, several Hematodinium sp. life stages were observed: trophonts, clump colonies, sporonts, arachnoid sporonts, sporoblasts and dinospores. Cultures initiated with sporonts progressed to motile dinospores; however, those initiated with amoeboid trophonts proliferated, but did not progress or formed schizont-like stages which were senescent artefacts. Plasmodial stages were associated with both trophonts and sporonts and could be differentiated by the presence of trichocysts on TEM. Macrodinospores were observed but not microdinospores; likely due to the low number of Hematodinium sp. cultures that progressed to the dinospore stage. No early life stages including motile filamentous trophonts or gorgonlocks were observed as previously noted in Hematodinium spp. from other crustacean hosts. All Hematodinium sp. life stages contained autofluorescent, membrane-bound electron dense granules that appeared to degranulate or be expelled from the cell during in vitro cultivation.

Sarcocystis neurona schizonts-associated encephalitis, chorioretinitis, and myositis in a two-month-old dog simulating toxoplasmosis, and presence of mature sarcocysts in muscles.

Sarcocystis neurona is an unusual species of the genus Sarcocystis. Opossums (Didelphis virginianus, D. albiventris) are the definitive hosts and several other species, including dogs, cats, marine mammals, and horses are intermediate or aberrant hosts. Sarcocysts are not known to form in aberrant hosts. Sarcocystis neurona causes fatal disease in horses (Equine Protozoal Myeloencephalitis, EPM). There are numerous reports of fatal EPM-like infections in other species, usually with central nervous system signs and associated with the schizont stage of S. neurona. Here, we report fatal disseminated S. neurona infection in a nine-week-old golden retriever dog from Mississippi, USA. Protozoal merozoites were identified in smears of the cerebrospinal fluid. Microscopically, lesions and protozoa were identified in eyes, tongue, heart, liver, intestines, nasal turbinates, skeletal muscle and brain, which reacted intensely with S. neurona polyclonal antibodies. Mature sarcocysts were seen in sections of muscles. These sarcocysts were ultrastructurally similar to those of S. neurona from experimentally infected animals. These data suggest that the dog is another intermediate host for S. neurona. Data suggest that the dog was transplacentally infected.

Eimeria arloingi: further studies on the development of some endogenous stages.

The present study investigated the ultrastructural characteristics of gamogony and oocyst wall formation of Eimeria arloingi in experimentally infected kids. The 18 newborn animals allocated to 3 equal groups. Two of groups, A, B were inoculated with a single dose of 1×10(3) and 1×10(5) sporulated oocysts of E. arloingi, respectively. At 7, 14, 21, 28, 35, and 42days postinoculation (DPI), 1 kid from each group was necropsied for ultrastructural studies. Transmission electron microscopy was used to screen for the presence of developmental stages of the parasite. All stages of microgametocyte and macrogametocyte developments and also oocyst wall formation were observed from 7 to 42DPI. Different stages of schizigony accompanied by marked proliferation of endoplasmic reticulum, mitochondria and several granular dividing nuclei were diagnosed in the affected epithelial cells. Young microgamonts were recognizable by an electron lucent parasitophorous vacuole and several dividing nuclei with prominent nucleolar and peripheral chromatin in the cytoplasm. At a later stage, the nuclei began to elongate and a single mitochondrion and two basal bodies were observed in close proximity nucleus. These bodies eventually protruded from the surface of the gametocyte and formed two flagellar structures. Up to 80-120 microgametes were produced per microgamont. Macrogamonts were recognized by the presence of wall-forming bodies of types 1 and 2. Electron lucent WFB2 appeared earlier than the electron denser WFB1 during the process of macrogametogenesis. The outer layer of the oocyst wall was formed by the release of the contents of WFB1 at the surface to form an electron dense layer. The WFB2 appeared, subsequently, to give rise to the electron lucent inner layer. WFB1 plays a major role in oocyst wall formation.

Features of autophagic cell death in Plasmodium liver-stage parasites.

Analyzing molecular determinants of Plasmodium parasite cell death is a promising approach for exploring new avenues in the fight against malaria. Three major forms of cell death (apoptosis, necrosis and autophagic cell death) have been described in multicellular organisms but which cell death processes exist in protozoa is still a matter of debate. Here we suggest that all three types of cell death occur in Plasmodium liver-stage parasites. Whereas typical molecular markers for apoptosis and necrosis have not been found in the genome of Plasmodium parasites, we identified genes coding for putative autophagy-marker proteins and thus concentrated on autophagic cell death. We characterized the Plasmodium berghei homolog of the prominent autophagy marker protein Atg8/LC3 and found that it localized to the apicoplast. A relocalization of PbAtg8 to autophagosome-like vesicles or vacuoles that appear in dying parasites was not, however, observed. This strongly suggests that the function of this protein in liver-stage parasites is restricted to apicoplast biology.

The haemosporidian parasites of bats with description of Sprattiella alecto gen. nov., sp. nov.

Four species of Haemoproteidae were found in Pteropus alecto Temminck, 1837 in Queensland, Australia: i) Johnsprentia copemani, Landau et al., 2012; ii) Sprattiella alecto gen. nov., sp. nov., characterised by schizonts in the renal vessels; iii) Hepatocystis levinei, Landau et al., 1985, originally described from Pteropus poliocephalus Temminck, 1825 and, experimentally from Culicoides nubeculosus and found in this new host and for which features of the hepatic schizonts are reported; iv) gametocytes of Hepatocystis sp. which are illustrated but cannot be assigned to a known species. A tentative interpretation of phylogenetic characters of haemosporidians of bats is provided from the morphology of the gametocytes and localisation of the tissue stages with respect to recent data on the phylogeny of bats.

Hepatozoon ursi n. sp. (Apicomplexa: Hepatozoidae) in Japanese black bear (Ursus thibetanus japonicus).

Morphological and genetic features of a new Hepatozoon species, Hepatozoon ursi n. sp., in Japanese black bear (Ursus thibetanus japonicus) were studied. Schizogonic developmental stages were observed in the lungs of Japanese black bears. The schizonts were sub-spherical in shape and 45.7+/-4.6 x 42.7+/-4.5 microm in size. Each mature schizont contained approximately 80-130 merozoites and 0-5 residual bodies. The merozoites were 7.0+/-0.7 x 1.8+/-0.3 microm in size. Intraleukocytic gametocytes were slightly curved, cigar-like in shape and had a beak-like protrusion at one end. The size of the gametocytes was 10.9+/-0.3 x 3.3+/-0.2 microm. The analyses of the18S rRNA gene sequences supported the hypothesis that H. ursi n. sp. is different from other Hepatozoon species. Mature Hepatozoon oocysts were detected in two species of ticks (Haemaphysalis japonica and Haemaphysalis flava) collected on the bears infected with H. ursi n. sp. Two measured oocysts were 263.2 x 234.0 microm and 331.8 x 231.7 microm, respectively. The oocysts contained approximately 40 and 50 sporocysts, respectively. The sporocysts were sub-spherical in shape and 31.2+/-2.5 x 27.0+/-2.9 microm in size. Each sporocyst contained at least 8-16 sporozoites, with the sporozoites being 12.2+/-1.4 x 3.5+/-0.5 microm in size. H. ursi n. sp. is the first Hepatozoon species recorded from the family Ursidae.

Four distinct pathways of hemoglobin uptake in the malaria parasite Plasmodium falciparum.

During the bloodstage of malaria infection, the parasite internalizes and degrades massive amounts of hemoglobin from the host red blood cell. Using serial thin-section electron microscopy and three-dimensional reconstruction, we demonstrate four independent, but partially overlapping, hemoglobin-uptake processes distinguishable temporally, morphologically, and pharmacologically. Early ring-stage parasites undergo a profound morphological transformation in which they fold, like a cup, onto themselves and in so doing take a large first gulp of host cell cytoplasm. This event, which we term the "Big Gulp," appears to be independent of actin polymerization and marks the first step in biogenesis of the parasite's lysosomal compartment-the food vacuole. A second, previously identified uptake process, uses the cytostome, a well characterized and morphologically distinct structure at the surface of the parasite. This process is more akin to classical endocytosis, giving rise to small (<0.004 fl) vesicles that are marked by the early endosomal regulatory protein Rab5a. A third process, also arising from cytostomes, creates long thin tubes previously termed cytostomal tubes in an actin-dependent manner. The fourth pathway, which we term phagotrophy, is similar to the Big Gulp in that it more closely resembles phagocytosis, except that phagotrophy does not require actin polymerization. Each of these four processes has aspects that are unique to Plasmodium, thus opening avenues to antimalarial therapy.