Acroeimeria lineri (McAllister, Upton, Freed, 1988) Paperna and Landsberg, 1989 in Mediterranean Geckos (Hemidactylus turcicus): Oocyst Morphometrics, Endogenous Developmental Stages, and Molecular Sequencing Support its Placement into Acroeimeria.

Between June 2016 and June 2019, we surveyed 62 Mediterranean geckos, Hemidactylus turcicus, from Abu Rawash, Giza, Egypt, for the presence of endoparasites. In June 2016, we found 3 individuals to be infected with Eimeria lineri. We studied the morphology and inner structures of its sporulated oocysts, and the locations of its intestinal endogenous stages. We also extracted genomic DNA from these sporulated oocysts and successfully sequenced a 632-bp fragment of the 18S rRNA gene. Phylogenetic analyses using this partial sequence allowed us to support previous studies that assigned E. lineri to the genus Acroeimeria. Our consensus sequence was used to query similar 18S rDNA sequences from GenBank, and 14 sequences were selected. The phylogenetic analysis inferred by maximum likelihood and Bayesian inference methods gave similar results, as both separated the sequences into 2 clades: (1) a monophyletic group of Goussia species (from fish); and (2) a strongly supported clade that separated 4 Choleoeimeria species from a polyphyletic group of species that clustered A. lineri with 3 other Acroeimeria species and 3 Eimeria species from lizards, including Eimeria tiliquae from Tiliqua rugosa (Gray, 1825), Eimeria tokayae from Gecko gecko (L., 1758), and Eimeria eutropidis from Eutropis macularia (Blyth, 1853). Our study supports the placement of E. lineri into the Acroeimeria and contributes additional life history information toward understanding the evolutionary origin of the Eimeria-like species that have sporocysts without Stieda bodies in their oocysts and that infect saurian reptiles. We also support the concept that several traits (morphological, endogenous, and gene sequences) are both necessary and important for authors to include when making generic reassignments within the eimeriid coccidia.

Molecular and morphological description of Sarcocystis kutkienae sp. nov. from the common raven (Corvus corax).

Until now, two Sarcocystis species, S. cornixi and S. corvusi, were known to employ members of the family Corvidae as intermediate hosts. Between 2013 and 2019, having examined leg muscles of 23 common ravens in Lithuania, sarcocysts were detected in 18 birds (78.3%). Using light microscopy, transmission electron microscopy (TEM), and molecular analysis (three genetic loci, 18S rDNA, 28S rDNA, and ITS1), sarcocysts found in the common raven were described as a new species S. kutkienae. Under a light microscope, the observed sarcocysts were ribbon-shaped (1500-8147 × 53-79 µm) and had a wavy striated cyst wall that reached up to 1.5 µm. Lancet-shaped bradyzoites were 7.7 × 2.2 µm (6.1-9.0 × 1.2-3.0 µm) in size. Ultrastructurally, the sarcocyst wall was 1.5-1.8 µm in thickness and had conical-like protrusions with minute invaginations of a parasitophorous vacuolar membrane. The cyst wall was type 1e-like. Limited genetic variability was observed between the 18S rDNA and 28S rDNA sequences of S. kutkienae and other Sarcocystis spp. using birds as intermediate hosts. In contrast, S. kutkienae could be clearly identified by comparing sequences. At this locus, sequences of S. kutkienae shared the highest similarity (89.5-89.7%) with those of S. cornixi. Phylogenetic analysis showed that S. kutkienae was most closely related to Sarcocystis spp. that employs birds as intermediate and definitive hosts. The issue relating to which species might serve as definitive hosts of S. kutkienae in Lithuania is addressed.

Gregarine Oocyst Production is a Function of Gametocyst Size.

Gregarine transmission depends upon the environmental encounter rate between viable infective oocysts and suitable hosts. Many factors determine the abundance and distribution of gregarine oocysts in the environment, but the primary factors are oocyst distribution, environmental persistence, and production rate. Prior studies have demonstrated factors affecting oocyst distribution and environmental persistence, but oocyst production rate is poorly understood. This study addresses the effects of gametocyst size on oocyst production. For each of 3 gregarine species, gametocyst size was determined, and the subsequent oocyst production of each gametocyst was quantified. Gregarine species with larger gametocysts produced more oocysts per gametocyst than species with smaller gametocysts. Likewise, within species, larger gametocysts produced more oocysts. The effect was stronger in larger gregarine species, probably as a reflection of the lower overall range of gametocyst size in the smaller gregarine species.

Ultrastructure of phagocytes and oocysts of Nematopsis sp. (Apicomplexa, Porosporidae) infecting Crassostrea rhizophorae in Northeastern Brazil / Ultraestrutura de fagócitos e oocistos de Nematopsis sp. (Apicomplexa, Porosporidae) infectando Crassostrea rhizophorae no Nordeste brasileiro

Abstract This work describes the detailed ultrastructural morphology of the phagocyte imprisoning an oyster of Nematopsis (Apicomplexa) found in Crassostrea rhizophorae, in the city of Maceió (AL), Brazil. The highly infected hosts had half-open leaflets with weak, slow retraction of the adductor muscles. Variable number of ellipsoid oocytes, either isolated and or clustered, was found between myofibrils of the adductor muscle. Each oocyst was incarcerated in a parasitophorous vacuole of host uninucleated phagocyte. The oocysts were composed of a dense wall containing a uninucleate vermiform sporozoite. The wall of the fine oocysts was composed of homogeneous electron-lucent material formed by three layers of equal thickness, having a circular orifice-micropyle obstructed by the operculum. The oocysts presented ellipsoid morphology with their wall was surrounded by a complex network of numerous microfibrils. Important details of the taxonomic value were visualized such as the ultrastructural organization of the oocyst wall and the organization of the micropyle and operculum, beyond the microfibrils that protrude from the oocyst wall only observed by transmission electron microscopy (TEM) and that may aid in the identification of the species. However, in order to clarify the systematic position of the species reported of the genus Nematopsis, it is important to proceed with genetic analyses.

Resumo Este trabalho descreve a morfologia ultraestrutural detalhada do fagócito encarcerando um oocisto de Nematopsis (Apicomplexa) encontrado em Crassostrea rhizophorae, na cidade de Maceió (AL), Brasil. Os hospedeiros muito infectados apresentavam valvas entreabertas com retração fraca e lenta dos músculos abdutores. Número variável de oócitos de forma elipsoide, isolados e ou agrupados foi encontrado entre as miofibrilas do músculo abdutor. Cada oocisto estava encarcerado num vacúolo parasitóforo do fagócito uninucleado do hospedeiro. Os oocistos eram compostos por uma parede densa contendo um esporozoíto vermiforme uninucleado. A parede dos oocistos finos era composta de material electron-lucente homogêneo formado por três camadas de espessura igual, possuindo um orifício circular - micrópila, obstruída pelo opérculo. Os oocistos apresentavam morfologia elipsoide, sua parede era circundada por uma complexa rede de numerosas microfibrilas. Detalhes de valor taxonômico importantes foram visualizados tais como: a organização ultraestrutural da parede do oocisto e a organização da micrópila e do opérculo, além das microfibrilas que se projetam da parede do oocisto, estrutura apenas observada em microscopia eletrônica de transmissão (MET) e que pode auxiliar na identificação da espécie. Contudo, para esclarecer a posição sistemática da maioria das espécies relatadas do gênero Nematopsis é importante prosseguir com as análises genéticas.

Ultrastructural analysis of Apicomplexa-Like parasites in two conch species Laevistrombus canarium and canarium urceus from Johor Straits, Malaysia.

The tropical conch, Laevistrombus canarium (Linnaeus, 1758) and Canarium urceus (Linneaus, 1758) are ecologically and economically important shellfish species in Malaysia and neighboring region. Their populations, however are currently declining and this histopathological study investigates the aspect of parasitism and diseases that may affect their well-being. Conch samples were randomly collected from their natural habitat and histological sections (4-5 µm) of various organs and tissues were examined under light microscope. This was followed by ultrastructure analysis on infected tissues using transmission electron microscope (TEM). Based on the histological analysis, large numbers of gamonts, sporocysts and trophozoites of Apicomplexa-like parasites were observed in the vacuolated cells and pyramidal crypt cells of the digestive tubules, and in the digestive ducts. Furthermore, coccidian and oocysts-like Pseudoklossia sp. stages were also observed in the cells of the kidney. Apart from that, spores with cyst-like structure were observed in the digestive gland and kidney. Although the parasites were present in most of the organs analyzed, there was no obvious symptom, inflammatory response or mortality incurred on both species, which implies the possibility of a non-virulent relationship like commensalisms or mutualism. However, more investigations, including molecular studies, are needed to confirm the parasite identification and dynamics, and to further evaluate the nature of relationship between Apicomplexa parasites and their host.

Sarcocystis jamaicensis n. sp., from Red-Tailed Hawks (Buteo jamaicensis) Definitive Host and IFN-γ Gene Knockout Mice as Experimental Intermediate Host.

Here, we report a new species of Sarcocystis with red-tailed hawk (RTH, Buteo jamaicensis) as the natural definitive host and IFN-γ gene knockout (KO) mice as an experimental intermediate host in which sarcocysts form in muscle. Two RTHs submitted to the Carolina Raptor Center, Huntersville, North Carolina, were euthanized because they could not be rehabilitated and released. Fully sporulated 12.5 × 9.9-µm sized sporocysts were found in intestinal scrapings of both hawks. Sporocysts were orally fed to laboratory-reared outbred Swiss Webster mice (SW, Mus musculus) and also to KO mice. The sporocysts were infective for KO mice but not for SW mice. All SW mice remained asymptomatic, and neither schizonts nor sarcocysts were found in any SW mice euthanized on days 54, 77, 103 (n = 2) or 137 post-inoculation (PI). The KO mice developed neurological signs and were necropsied between 52 to 68 days PI. Schizonts/merozoites were found in all KO mice euthanized on days 52, 55 (n = 3), 59, 61 (n = 2), 66, and 68 PI and they were confined to the brain. The predominant lesion was meningoencephalitis characterized by perivascular cuffs, granulomas, and necrosis of the neural tissue. The schizonts/merozoites were located in neural tissue and were apparently extravascular. Brain homogenates from infected KO mice were infective to KO mice by subcutaneous inoculation and when seeded on to CV-1 cells. Microscopic sarcocysts were found in skeletal muscles of 5 of 8 KO mice euthanized between 55-61 days PI. Only a few sarcocysts were detected. Sarcocysts were microscopic, up to 3.5 mm long. When viewed with light microscopy, the sarcocyst wall appeared thin (<1 µm thick) and smooth. By transmission electron microscopy, the sarcocyst wall classified as "type 1j" (new designation). Molecular characterization using 18S rRNA, 28S rRNA, ITS-1, and cox1 genes revealed a close relationship with Sarcocystis microti and Sarcocystis glareoli; both species infect birds as definitive hosts. The parasite in the present study was biologically and molecularly different from species so far described in RTHs and we therefore propose a new species name, Sarcocystis jamaicensis n. sp.

Macrophages facilitate the excystation and differentiation of Toxoplasma gondii sporozoites into tachyzoites following oocyst internalisation.

Toxoplasma gondii is a common parasite of humans and animals, which is transmitted via oocysts in cat faeces or tissue cysts in contaminated meat. The robust oocyst and sporocyst walls protect the infective sporozoites from deleterious external attacks including disinfectants. Upon oocyst acquisition, these walls lose their integrity to let the sporozoites excyst and invade host cells following a process that remains poorly understood. Given the resistance of the oocyst wall to digestive enzymes and the ability of oocysts to cause parenteral infections, the present study investigated the possible contribution of macrophages in supporting sporozoite excystation following oocyst internalisation. By using single cell micromanipulations, real-time and time-point imaging techniques, we demonstrated that RAW macrophages could interact rapidly with oocysts and engulfed them by remodelling of their actin cytoskeleton. Internalised oocysts were associated to macrophage acidic compartments and showed evidences of wall disruption. Sporozoites were observed in macrophages containing oocyst remnants or in new macrophages, giving rise to dividing tachyzoites. All together, these results highlight an unexpected role of phagocytic cells in processing T. gondii oocysts, in line with non-classical routes of infection, and open new perspectives to identify chemical factors that lead to oocyst wall disruption under physiological conditions.

Molecular Characterization and Immune Protection of a New Conserved Hypothetical Protein of Eimeria tenella.

The genome sequences of Eimeria tenella have been sequenced, but >70% of these genes are currently categorized as having an unknown function or annotated as conserved hypothetical proteins, and few of them have been studied. In the present study, a conserved hypothetical protein gene of E. tenella, designated EtCHP559, was cloned using rapid amplification of cDNA 5'-ends (5'RACE) based on the expressed sequence tag (EST). The 1746-bp full-length cDNA of EtCHP559 contained a 1224-bp open reading frame (ORF) that encoded a 407-amino acid polypeptide with the predicted molecular weight of 46.04 kDa. Real-time quantitative PCR analysis revealed that EtCHP559 was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts and second generation merozoites). The ORF was inserted into pCold-TF to produce recombinant EtCHP559. Using western blotting, the recombinant protein was successfully recognized by rabbit serum against E. tenella sporozoites. Immunolocalization by using EtCHP559 antibody showed that EtCHP559 was mainly distributed on the parasite surface in free sporozoites and became concentrated in the anterior region after sporozoites were incubated in complete medium. The EtCHP559 became uniformly dispersed in immature and mature schizonts. Inhibition of EtCHP559 function using anti-rEtCHP559 polyclonal antibody reduced the ability of E. tenella sporozoites to invade host cells by >70%. Animal challenge experiments demonstrated that the recombinant EtCHP559 significantly increased the average body weight gain, reduced the oocyst outputs, alleviated cecal lesions of the infected chickens, and resulted in anticoccidial index >160 against E. tenella. These results suggest that EtCHP559 plays an important role in sporozoite invasion and could be an effective candidate for the development of a new vaccine against E. tenella.

Reclassification of Eimeria pogonae Walden (2009) as Choleoeimeria pogonae comb. nov. (Apicomplexa: Eimeriidae).

The presented paper provides a reclassification of Eimeria pogonae from Pogona vitticeps into the correct genus Choleoeimeria. A description of exogenous and endogenous stages of biliary coccidium is given. Sporulation of the oocysts was endogenous. The mature oocysts contained four sporocysts each with two sporozoites. Oocysts were ellipsoidal in shape, with average length/width ratio 1.7 and measured 28.4 (SD1.5) × 16.8 (SD 1.5). The micropyle, residuum, and polar granules were absent from the sporulated oocysts. Ovoidal in shape, sporosysts without Steida bodies contained residuum and two elongated and boat-shaped sporozoites. The endogenous stages of the coccidia were located mainly in the epithelium of bile ducts; however, single-epithelium cells of the gallbladder were also infected.

Atomic force microscopic imaging of Acanthamoeba castellanii and Balamuthia mandrillaris trophozoites and cysts.

Light microscopy and electron microscopy have been successfully used in the study of microbes, as well as free-living protists. Unlike light microscopy, which enables us to observe living organisms or the electron microscope which provides a two-dimensional image, atomic force microscopy provides a three-dimensional surface profile. Here, we observed two free-living amoebae, Acanthamoeba castellanii and Balamuthia mandrillaris under the phase contrast inverted microscope, transmission electron microscope and atomic force microscope. Although light microscopy was of lower magnification, it revealed functional biology of live amoebae such as motility and osmoregulation using contractile vacuoles of the trophozoite stage, but it is of limited value in defining the cyst stage. In contrast, transmission electron microscopy showed significantly greater magnification and resolution to reveal the ultra-structural features of trophozoites and cysts including intracellular organelles and cyst wall characteristics but it only produced a snapshot in time of a dead amoeba cell. Atomic force microscopy produced three-dimensional images providing detailed topographic description of shape and surface, phase imaging measuring boundary stiffness, and amplitude measurements including width, height and length of A. castellanii and B. mandrillaris trophozoites and cysts. These results demonstrate the importance of the application of various microscopic methods in the biological and structural characterization of the whole cell, ultra-structural features, as well as surface components and cytoskeleton of protist pathogens.

Protomagalhaensia richardsoni n. sp. (Apicomplexa: Eugregarinida: Blabericolidae): a new gregarine parasitizing the giant lobster cockroach, Henschoutedenia flexivitta (Dictyoptera: Blaberidae).

Protomagalhaensia richardsoni n. sp. (Apicomplexa: Eugregarinida: Blabericolidae) is described from the giant lobster cockroach, Henschoutedenia flexivitta (Dictyoptera: Blattaria: Blaberidae: Oxyhaloinae: Nauphoetini). Oocysts within the genus are dolioform with polar plates. Those of Protomagalhaensia granulosae, Protomagalhaensia wolfi, and Protomagalhaensia blaberae possess distinct apical spines and a sagittal depression that are absent or reduced in P. richardsoni and Protomagalhaensia cerastes. Oocysts of P. richardsoni are significantly longer with larger sporozoite-bearing cavities than those of P. blaberae, P. cerastes, P. granulosae, and P. wolfi (external oocyst length 8.07 µm vs. 7.42 µm, 7.50 µm, 6.87 µm, 7.56 µm, respectively; internal oocyst length 6.94 µm vs. 6.44 µm, 6.77 µm, 6.09 µm, 6.72 µm, respectively). All 5 species are also distinguished by unique oocyst length/width ratios. No unique morphological structure distinguishes among the gametocysts of Protomagalhaensia species, but gametocysts of P. richardsoni are significantly shorter than those of P. blaberae, P. cerastes, P. granulosae, and P. wolfi (gametocyst length 184.3 µm vs. 325.15 µm, 253.27 µm, 273.63 µm, 218.3 µm, respectively). No structurally unique morphological gamont feature distinguishes among species of Protomagalhaensia. Rather, species distinctions are morphometric in nature. In general, gamonts of P. richardsoni are readily distinguished from those of P. cerastes and P. wolfi based on size alone: the latter species being roughly half the size of P. richardsoni. Gamonts of P. richardsoni are most similar to those of P. granulosae and P. blaberae but with relatively smaller primites and more slender satellites.

ß-1,3-glucan, which can be targeted by drugs, forms a trabecular scaffold in the oocyst walls of Toxoplasma and Eimeria.

UNLABELLED: The walls of infectious pathogens, which are essential for transmission, pathogenesis, and diagnosis, contain sugar polymers that are defining structural features, e.g., ß-1,3-glucan and chitin in fungi, chitin in Entamoeba cysts, ß-1,3-GalNAc in Giardia cysts, and peptidoglycans in bacteria. The goal here was to determine in which of three walled forms of Toxoplasma gondii (oocyst, sporocyst, or tissue cyst) is ß-1,3-glucan, the product of glucan synthases and glucan hydrolases predicted by whole-genome sequences of the parasite. The three most important discoveries were as follows. (i) ß-1,3-glucan is present in oocyst walls of Toxoplasma and Eimeria (a chicken parasite that is a model for intestinal stages of Toxoplasma) but is absent from sporocyst and tissue cyst walls. (ii) Fibrils of ß-1,3-glucan are part of a trabecular scaffold in the inner layer of the oocyst wall, which also includes a glucan hydrolase that has a novel glucan-binding domain. (iii) Echinocandins, which target the glucan synthase and kill fungi, arrest development of the Eimeria oocyst wall and prevent release of the parasites into the intestinal lumen. In summary, ß-1,3-glucan, which can be targeted by drugs, is an important component of oocyst walls of Toxoplasma but is not a component of sporocyst and tissue cyst walls. IMPORTANCE: We show here that walls of Toxoplasma oocysts, the infectious stage shed by cats, contain ß-1,3-glucan, a sugar polymer that is a major component of fungal walls. In contrast to fungi, ß-1,3-glucan is part of a trabecular scaffold in the inner layer of the oocyst wall that is independent of the permeability barrier formed by the outer layer of the wall. While glucan synthase inhibitors kill fungi, these inhibitors arrest the development of the oocyst walls of Eimeria (an important chicken pathogen that is a surrogate for Toxoplasma) and block release of oocysts into the intestinal lumen. The absence of ß-1,3-glucan in tissue cysts of Toxoplasma suggests that drugs targeted at the glucan synthase might be used to treat Eimeria in chickens but not to treat Toxoplasma in people.

Didelphis aurita (Marsupialia: Didelphidae): a new host for Sarcocystis lindsayi (Apicomplexa: Sarcocystidae).

Nine opossums, Didelphis aurita , were captured in the city of Seropédica, State of Rio de Janeiro, Brazil and examined for species of Sarcocystis. Sporocysts were observed in the mucosal scrapings of the small intestine from 3 opossums. Five budgerigars, Melopsittacus undulatus , were infected with sporocysts from each of these infected opossums and 5 budgerigars were used as controls. Of the 15 sporocyst-treated budgerigars, 5 birds that received sporocysts from 1 of the infected opossums developed tissue parasites. Meronts in the vascular endothelium of the lung venous capillaries and cysts in the skeletal and cardiac muscle cells were observed in histological sections stained with hematoxylin and eosin. The microscopic cysts, which were predominantly in the tongue and leg muscles, ranged from 65.3 to 118.1 µm in length and 14.0 to 29.4 µm in width and from 0.9 to 1.9 µm in thickness of the cystic wall. Sections examined by transmission electron microscopy revealed that the cyst wall contained numerous slender and jagged-shaped protrusions, each with a finger-like formation at the end. The morphology, especially of the cyst wall, and the morphometry of the tissue cysts indicate that the parasite is Sarcocystis lindsayi and, therefore, the opossum, D. aurita , is now considered a definitive host for this species in Brazil.

Movable computer ruler (MCR): a new method for measuring the size of Toxoplasma gondii cysts, tachyzoites and other selected parasites.

A new method for measuring the size of parasites and other objects using optical microscopy was developed using a specifically designed movable computer ruler (MCR) derived from digital images of a stage micrometer. Subsequently, MCR can be superimposed on images of parasites to measure their size. MCR derived from the stage micrometer under a particular objective lens can be used to measure the size of an object acquired by the same lens/microscope/camera system. The conditions are fixed for every superimposed image including width, height, pixel number and density. The MCR was tested using selected parasites, and shown to be as accurate as the ocular micrometer disk, screw micrometer eyepiece and image analysis software. The lower technical complexity of the MCR method makes it applicable even in laboratories with limited resources.

Isospora riyadhensis n. sp. (Apicomplexa: Eimeriidae) from the worm lizard Diplometopon zarudnyi Nikolskii (Amphisbaenia: Trogonophidae) in Saudi Arabia.

Isospora riyadhensis n. sp. is described from the intestine of the worm lizard Diplometopon zarudnyi Nikolskii in Saudi Arabia, where its prevalence was 26.6%. Its oöcysts are spherical to subspherical and measure 23 × 20 µm. The sporocysts, which are tetrazoic and ovoid, measure 13 × 8 µm, whereas their sporozoites are banana-shaped, have anterior and posterior refractile bodies and measure 12 × 3 µm. Oöcysts are passed unsporulated, and the majority become fully sporulated within 3 days at 25 ° C. All endogenous stages develop in the cytoplasm of epithelial cells in the posterior region of the small intestine, from where meronts, microgamonts and macrogamonts are described.

The conformation and activation of Fyn kinase in the oocyte determine its localisation to the spindle poles and cleavage furrow.

Several lines of evidence imply the involvement of Fyn, a Src family kinase, in cell-cycle control and cytoskeleton organisation in somatic cells. By live cell confocal imaging of immunostained or cRNA-microinjected mouse oocytes at metaphase of the second meiotic division, membrane localisation of active and non-active Fyn was demonstrated. However, Fyn with a disrupted membrane-binding domain at its N-terminus was targeted to the cytoplasm and spindle in its non-active form and concentrated at the spindle poles when active. During metaphase exit, the amount of phosphorylated Fyn and of spindle-poles Fyn decreased and it started appearing at the membrane area of the cleavage furrow surrounding the spindle midzone, either asymmetrically during polar body II extrusion or symmetrically during mitosis. These results demonstrate that post-translational modifications of Fyn, probably palmitoylation, determine its localisation and function; localisation of de-palmitoylated active Fyn to the spindle poles is involved in spindle pole integrity during metaphase, whereas the localisation of N-terminus palmitoylated Fyn at the membrane near the cleavage furrow indicates its participation in furrow ingression during cytokinesis.

Molecular characterisation of a novel family of cysteine-rich proteins of Toxoplasma gondii and ultrastructural evidence of oocyst wall localisation.

Among apicomplexan parasites, the coccidia and Cryptosporidium spp. are important pathogens of livestock and humans, and the environmentally resistant stage (oocyst) is essential for their transmission. Little is known of the chemical and molecular composition of the oocyst wall. Currently, the only parasite molecules shown to be involved in oocyst wall formation are the tyrosine-rich proteins gam56, gam82 and gam230 of Eimeria spp. and the cysteine-rich proteins COWP1 and COWP8 of Cryptosporidium parvum. In the present study, we searched the ToxoDB database for the presence of putative Toxoplasma gondii oocyst wall proteins (OWPs) and identified seven candidates, herein named TgOWP1 through TgOWP7, showing homology to the Cryptosporidium COWPs. We analysed a cDNA library from partially sporulated oocysts of T. gondii and cloned the full-length cDNAs encoding TgOWP1, TgOWP2 and TgOWP3, which consist of 499, 462 and 640 amino acids, respectively. The three proteins share 24% sequence identity with each other and a markedly similar overall structure, based on the presence of an N-terminal leader peptide followed by tandem duplications of a six-cysteine amino acid motif closely related to the Type I repeat of COWPs. Using antisera to recombinant TgOWP1, TgOWP2 and TgOWP3, we showed by Western blot that these molecules are expressed in T. gondii oocysts but are not detectable in tachyzoites. The solubilisation of TgOWP1-3 strictly depended on the presence of reducing agents, consistent with a likely involvement of these proteins in multimeric complexes mediated by disulphide bridges. Immunofluorescence analysis allowed the localisation of TgOWP1, TgOWP2 and TgOWP3 to the oocyst wall. Additionally, using immunoelectron microscopy and the 1G12 monoclonal antibody, TgOWP3 was specifically detected in the outer layer of the oocyst wall, thus representing the first validated molecular marker of this structure in T. gondii.

Primeiro relato de Cryptosporidium spp: em emas (Rhea americana) cativas de zoológico no Brasil / First report of Cryptosporidium spp: in captive rheas (Rhea americana) in a Brazilian zoo

Este estudo objetivou avahar a ocorrência de oocistos de Cryptosporidium spp. em emas (Rhea americana) cativas no Parque Zoológico da Fundação Zoobotânica do Rio Grande do Sul, sul do Brasil. Foram identificados oocistos de Cryptosporidium spp. em esfregaços de fezes de emas e em amostras de agua, coradas pela técnica de Ziehl-Neelsen modificada. A análise morfométrica dos coccídeos revelou pequenos oocistos esféricos medindo, em media, 4,91 um X 4,91 /im e relação comprimento/largura de razão 1. A confirmação da prêsera de oocistos nas amostras de fezes e de agua é relevante pelo potencial de transmissão e manutenção da criptosporidiose em hospedeiros susceptíveis. Este é o primeiro relato de Cryptosporidium spp. em R. americana no Brasil.

The aim of this study was to evaluate the occurrence of Cryptosporidium spp. oocysts in common rhea (Rhea americana) living in captivity in the zoological park of the Zoobotanical Foundation of the State of Rio Grande do Sul, southern Brazil. Cryptosporidium spp. oocysts were detected in fecal smears of common rhea and in water samples by using the modified Ziehl-Neelsen staining method. The morphometric analysis of coccidia revealed small spherical oocysts, measuring, on average, 4.91 [im x 4.91 fim, and a length/width ratio of 1. The detection of Cryptosporidium spp. oocysts in fecal and water samples is important as it can indicate the transmission and maintenance of cryptosporidiosis in susceptible hosts. This is the first report of Cryptosporidium spp. in R. americana in Brazil.

Life cycle of Hepatozoon canis (Apicomplexa: Adeleorina: Hepatozoidae) in the tick Rhipicephalus sanguineus and domestic dog (Canis familiaris).

The life cycle of the apicomplexan protozoon Hepatozoon canis in its natural hosts Rhipicephalus sanguineus (tick) and Canis familiaris (domestic dog) was studied in an experimental infection. Tick nymphs were fed on a naturally infected dog, or they were infected by percutaneous injection of blood. Dogs were inoculated by ingestion of adult ticks containing mature oocysts. Gamonts were in syzygy 24 hr after percutaneous injection of ticks. Early oocysts were detected 96 hr after nymph repletion, and mature oocysts in adult ticks were infective to dogs 40 days postmolt. Merogony was detected in dog bone marrow from 13 days postinoculation (PI) and included meronts containing 20-30 micromerozoites, and a second type with 2-4 macromerozoites. Monozoic cysts were observed in the spleen in conjunction with merogony. Gamontogony with infection of leukocytes by micromerozoites occurred from 26 days PI, and gamont parasitemia, which completed the life cycle, was detected 28 days PI. The length of the life cycle from nymphal attachment to parasitemia in dogs was 81 days. Increased body temperatures were evident from 16 to 27 days PI and paralleled the time of intensive bone marrow merogony. Skeletal pain and recumbency were manifested in 2 dogs. This study further elucidates the life cycle of H. canis and provides a sequential morphologic description of H. canis merogony, gamontogony, and sporogony.

Ultrastructural review of Choleoeimeria spp., a coccidium infecting the gall-bladder epithelium of reptiles.

Choleoeimeria Paperna and Landsberg, 1989 is a reptile coccidium with unique features. Its endogenous development occurs in the cells of the bile epithelium. Its host cell while becoming hypertrophic emerges above the epithelial surface. The following species studied by electron microscopy: C. alloagamae Paperna, 2007 from Agama sp. West Africa; C. allogehyrae Paperna, 2007 from Gehyra australis and C. heteronotis Paperna, 2007 from Heteronotia binoei, from Australia, and C. pachydactyli Paperna and Landsberg, 1989 from Pachydactylus capensis from South Africa. The fine structure of the respective endogenous stages is fairly uniform. The host-cell hypertrophy coincides with a drastic depletion of the microvilli, their junction zone with the underlying cell extends into numerous long and fine membranal out-folds. The PV of all infected cells is filled with typical round granular particles. Young meronts undergo binary fission. The differentiating microgamont develops an expanded multilobed body. Macrogamont's organelles include type 1 and type 2 wall forming bodies, canaliculi and granular bodies, suspected to be the precursors of the sporozoites refractile bodies. The oocyst wall forms from 4 wall-membranes consolidating over the zygote plasmalemma.