GAMOGONY OF SARCOCYSTIS STRIXI IN MAMMALIAN CELL CULTURES.

We are interested in the disease ecology of Sarcocystis species that infect birds of prey as definitive and intermediate hosts. The present study was done to test our hypothesis that a laboratory model can be developed for sarcocystis infection in mammals using gamma interferon gene knockout (KO) mice as a source of Sarcocystis strixi bradyzoites and mammalian cell cultures as a source of sporulated S. strixi oocysts. Sporocysts of S. strixi from a naturally infected barred owl (Strix varia) were fed to KO mice to produce sarcocysts, and the enclosed bradyzoites were obtained by acid-pepsin digestion of abdominal and thigh muscles. Bradyzoites, metrocytes, and an unusual spherical stage were seen in digest before the inoculation of host cells. The spherical stages stained dark with Giemsa stain, but no nucleus was observed, and they were seen free and associated with the concave portion of some bradyzoites. Examination of infected cell cultures demonstrated that macrogamonts and microgamonts were present at 24 hr post-inoculation. Since sporulated oocysts were not observed, we had to reject our current hypothesis.

Assessing viability and infectivity of foodborne and waterborne stages (cysts/oocysts) of Giardia duodenalis, Cryptosporidium spp., and Toxoplasma gondii: a review of methods.

Giardia duodenalis, Cryptosporidium spp. and Toxoplasma gondii are protozoan parasites that have been highlighted as emerging foodborne pathogens by the Food and Agriculture Organization of the United Nations and the World Health Organization. According to the European Food Safety Authority, 4786 foodborne and waterborne outbreaks were reported in Europe in 2016, of which 0.4% were attributed to parasites including Cryptosporidium, Giardia and Trichinella. Until 2016, no standardized methods were available to detect Giardia, Cryptosporidium and Toxoplasma (oo)cysts in food. Therefore, no regulation exists regarding these biohazards. Nevertheless, considering their low infective dose, ingestion of foodstuffs contaminated by low quantities of these three parasites can lead to human infection. To evaluate the risk of protozoan parasites in food, efforts must be made towards exposure assessment to estimate the contamination along the food chain, from raw products to consumers. This requires determining: (i) the occurrence of infective protozoan (oo)cysts in foods, and (ii) the efficacy of control measures to eliminate this contamination. In order to conduct such assessments, methods for identification of viable (i.e. live) and infective parasites are required. This review describes the methods currently available to evaluate infectivity and viability of G. duodenalis cysts, Cryptosporidium spp. and T. gondii oocysts, and their potential for application in exposure assessment to determine the presence of the infective protozoa and/or to characterize the efficacy of control measures. Advantages and limits of each method are highlighted and an analytical strategy is proposed to assess exposure to these protozoa.

TITLE: Estimation de la viabilité et infectiosité des stades (kystes et oocystes) de Giardia duodenalis, Cryptosporidium spp. et Toxoplasma gondii transmis par la nourriture et l'eau : une revue des méthodes. ABSTRACT: Giardia duodenalis, Cryptosporidium spp. et Toxoplasma gondii sont des parasites protozoaires qui ont été soulignés comme agents pathogènes émergents dans les aliments par l'Organisation des Nations Unies pour l'alimentation et l'agriculture et l'Organisation Mondiale de la Santé. Selon l'Autorité Européenne de Sécurité des Aliments, 4786 épidémies d'origine alimentaire et hydrique ont été enregistrées en Europe en 2016, dont 0.4% ont été attribuées à des parasites, incluant Cryptosporidium, Giardia et Trichinella. Jusqu'en 2016, aucune méthode standardisée n'était disponible pour détecter les kystes de Giardia et les oocystes de Cryptosporidium et Toxoplasma dans les aliments. Aucune réglementation n'est donc proposée concernant ces dangers. Cependant, compte tenu de leur faible dose infectieuse, l'ingestion d'une quantité d'aliments faiblement contaminés peut entraîner une infection de l'homme. Pour évaluer le risque lié aux protozoaires dans les aliments, des efforts doivent être faits dans l'évaluation de l'exposition pour estimer la contamination le long de la chaîne alimentaire, depuis la matière première jusqu'aux consommateurs. Cette évaluation nécessite de déterminer : (i) la prévalence de parasites infectieux dans les aliments, (ii) l'efficacité des mesures de maîtrise pour éliminer cette contamination. Pour mener une telle évaluation, des méthodes capables d'identifier des parasites viables (vivants) et infectieux sont requises. Cette revue décrit les méthodes actuellement disponibles permettant d'évaluer l'infectiosité et la viabilité des kystes de G. duodenalis et des oocystes de Cryptosporidium spp. et T. gondii, et leur potentiel pour être appliquées dans l'évaluation de l'exposition pour déterminer la présence de parasites infectieux et/ou caractériser l'efficacité des mesures de maîtrise. Les avantages et limites de chaque méthode sont présentés et une stratégie d'analyses est proposée pour évaluer l'exposition à ces protozoaires.

A cell culture platform for Cryptosporidium that enables long-term cultivation and new tools for the systematic investigation of its biology.

Cryptosporidium parasites are a major cause of diarrhoea that pose a particular threat to children in developing areas and immunocompromised individuals. Curative therapies and vaccines are lacking, mainly due to lack of a long-term culturing system of this parasite. Here, we show that COLO-680N cells infected with two different Cryptosporidium parvum strains produce sufficient infectious oocysts to infect subsequent cultures, showing a substantial fold increase in production, depending on the experiment, over the most optimistic HCT-8 models. Oocyst identity was confirmed using a variety of microscopic- and molecular-based methods. This culturing system will accelerate research on Cryptosporidium and the development of anti-Cryptosporidium drugs.

Mammalian cellular culture models of Trypanosoma cruzi infection: a review of the published literature.

Cellular culture infection with Trypanosoma cruzi is a tool used to dissect the biological mechanisms behind Chagas disease as well as to screen potential trypanocidal compounds. Data on these models are highly heterogeneous, which represents a challenge when attempting to compare different studies. The purpose of this review is to provide an overview of the cell culture infectivity assays performed to date. Scientific journal databases were searched for articles in which cultured cells were infected with any Trypanosoma cruzi strain or isolate regardless of the study's goal. From these articles the cell type, parasite genotype, culture conditions and infectivity results were extracted. This review represents an initial step toward the unification of infectivity model data. Important differences were detected when comparing the pathophysiology of Chagas disease with the experimental conditions used in the analyzed studies. While Trypanosoma cruzi preferentially infects stromal cells in vivo, most of the assays employ epithelial cell lines. Furthermore, the most commonly used parasite strain (Tulahuen-TcVI) is associated with chagasic cardiomyopathy only in the Southern Cone of South America. Suggestions to overcome these discrepancies include the use of stromal cell lines and parasite genotypes associated with the known characteristics of the natural history of Chagas disease.

The toxoplasma-host cell junction is anchored to the cell cortex to sustain parasite invasive force.

BACKGROUND: The public health threats imposed by toxoplasmosis worldwide and by malaria in sub-Saharan countries are directly associated with the capacity of their related causative agents Toxoplasma and Plasmodium, respectively, to colonize and expand inside host cells. Therefore, deciphering how these two Apicomplexan protozoan parasites access their host cells has been highlighted as a priority research with the perspective of designing anti-invasive molecules to prevent diseases. Central to the mechanism of invasion for both genera is mechanical force, which is thought to be applied by the parasite at the interface between the two cells following assembly of a unique cell-cell junction but this model lacks direct evidence and has been challenged by recent genetic studies. In this work, using parasites expressing the fluorescent core component of this junction, we analyze characteristic features of the kinematics of penetration of more than 1,000 invasion events. RESULTS: The majority of invasion events occur with a typical forward rotational progression of the parasite through a static junction into an invaginating host cell plasma membrane. However, if parasites encounter resistance and if the junction is not strongly anchored to the host cell cortex, as when parasites do not secrete the toxofilin protein and, therefore, are unable to locally remodel the cortical actin cytoskeleton, the junction travels retrogradely with the host cell membrane along the parasite surface allowing the formation of a functional vacuole. Kinetic measurements of the invasive trajectories strongly support a similar parasite driven force in both static and capped junctions, both of which lead to successful invasion. However, about 20% of toxofilin mutants fail to enter and eventually disengage from the host cell membrane while the secreted RhOptry Neck (RON2) molecules are posteriorally capped before being cleaved and released in the medium. By contrast in cells characterized by low cortex tension and high cortical actin dynamics junction capping and entry failure are drastically reduced. CONCLUSIONS: This kinematic analysis newly highlights that to invade cells parasites need to engage their motor with the junction molecular complex where force is efficiently applied only upon proper anchorage to the host cell membrane and cortex.

Molecular basis of mammalian cell invasion by Trypanosoma cruzi.

Establishment of infection by Trypanosoma cruzi, the agent of Chagas' disease, depends on a series of events involving interactions of diverse parasite molecules with host components. Here we focus on the mechanisms of target cell invasion by metacyclic trypomastigotes (MT) and mammalian tissue culture trypomastigotes (TCT). During MT or TCT internalization, signal transduction pathways are activated both in the parasite and the target cell, leading to Ca2+ mobilization. For cell adhesion, MT engage surface glycoproteins, such as gp82 and gp35/50, which are Ca2+ signal-inducing molecules. In T. cruzi isolates that enter host cells in gp82-mediated manner, parasite protein tyrosine kinase as well as phospholipase C are activated, and Ca2+ is released from I P3-sensitive stores, whereas in T. cruzi isolates that attach to target cells mainly through gp35/50, the signaling pathway involving adenylate cyclase appears to be stimulated, with Ca2+ release from acidocalciosomes. In addition, T. cruzi isolate-dependent inhibitory signals, mediated by MT-specific gp90, may be triggered both in the host cell and the parasite. The repertoire of TCT molecules implicated in cell invasion includes surface glycoproteins of gp85 family, with members containing binding sites for laminin and cytokeratin 18, enzymes such as cruzipain, trans-sialidase, and an oligopeptidase B that generates a Ca2+-agonist from a precursor molecule.

Molecular basis of mammalian cell invasion by Trypanosoma cruzi

O estabelecimento da infecção por Trypanosoma cruzi, o agente da doença de Chagas, depende de uma série de eventos envolvendo interações de diversas moléculas do parasita com componentes do hospedeiro. Focalizamos aqui os mecanismos de invasão celular por tripomastigotas metacíclicos (TM) e por tripomastigotas de cultura de tecido (TCT). Durante a internalização de TM ou TCT, vias de transdução de sinal são ativadas tanto no parasita como na célula alvo, acarretando a mobilização de Ca2+. Para adesão, TM utiliza as glicoproteínas de superfície como a gp82 e gp35/50, que são moléculas indutoras de sinal de Ca2+. Em isolados de T. cruzi que entram na célula hospedeira de maneira dependente de gp82, a proteína tirosina quinase assim como a fosfolipase C do parasita são ativadas, e Ca2+ é liberado de reservatórios sensíveis a IP3, enquanto em isolados de T. cruzi que se ligam às células alvo através de gp35/50, a via de sinalização envolvendo adenilil ciclase parece ser estimulada, com liberação de Ca2+ de acidocalciossomos. Além disso, dependendo do isolado de T. cruzi, sinais inibitórios mediados por gp90 específica de TM podem ser desencadeados tanto na célula hospedeira como no parasita. O repertório de moléculas de TCT implicadas na invasão celular inclui glicoproteínas de superfície da família gp85, com membros contendo sitos de ligação à laminina e citoqueratina 18, enzimas como a cruzipaína, trans-sialidase, e uma oligopeptidase B que gera um agonista de Ca2+ a partir de uma molécula precursora. .

Generally applicable methods to purify intracellular coccidia from cell cultures and to quantify purification efficacy using quantitative PCR.

The objective of this study was to evaluate the utility of a simple, efficient, and rapid method for the isolation of Sarcocystis neurona merozoites and Besnoitia darlingi tachyzoites from cultured cells. The efficacy of this purification method was assessed by microscopy, SDS-PAGE, Western blotting, immuno-fluorescence, and three novel quantitative PCR assays. Culture medium containing host cell debris and parasites was eluted through PD-10 desalting columns. This purification method was compared to alternatives employing filtration through a cellulose filter pad or filter paper. The estimated recovery of S. neurona merozoites purified by the column method was 82% (+/-3.7) of the original merozoites with 97.5% purity. In contrast, estimated recovery of S. neurona merozoites purified by filter pad and filter paper was 40% and 30% with 76% and 83% purity, respectively. The same procedures were applied to purify B. darlingi tachyzoites from cultured cells. Of the original cultured B. darlingi tachyzoites, 94% (+/-2.5) were recovered from the PD-10 column with 96.5%, purity whereas percentage recovery of B. darlingi tachyzoites purified by filter pad and filter paper were 51% and 35% with 84% and 88% purity, respectively. All described methods maintained sterility so that purified parasites could be subsequently cultured in vitro. However, purification using a PD-10 column minimized parasite loss and the loss of viability as determined by the trypan blue dye exclusion assay, the rate of parasite production, and plaque forming efficiency in cell culture. Moreover, column-purified parasites improved the sensitivity of an immuno-fluorescent (IFA) analysis and real-time quantitative PCR assays targeted to parasite 18S ribosomal DNA and hsp70 genes. This technique appears generally applicable for purifying coccidia grown in cell cultures.

Mammalian cell invasion and intracellular trafficking by Trypanosoma cruzi infective forms.

Trypanosoma cruzi, the etiological agent of Chagas disease, occurs as different strains or isolates that may be grouped in two major phylogenetic lineages: T. cruzi I, associated with the sylvatic cycle and T. cruzi II, linked to the human disease. In the mammalian host the parasite has to invade cells and many studies implicated the flagellated trypomastigotes in this process. Several parasite surface components and some of host cell receptors with which they interact have been identified. Our work focused on how amastigotes, usually found growing in the cytoplasm, can invade mammalian cells with infectivities comparable to that of trypomastigotes. We found differences in cellular responses induced by amastigotes and trypomastigotes regarding cytoskeletal components and actin-rich projections. Extracellularly generated amastigotes of T. cruzi I strains may display greater infectivity than metacyclic trypomastigotes towards cultured cell lines as well as target cells that have modified expression of different classes of cellular components. Cultured host cells harboring the bacterium Coxiella burnetii allowed us to gain new insights into the trafficking properties of the different infective forms of T. cruzi, disclosing unexpected requirements for the parasite to transit between the parasitophorous vacuole to its final destination in the host cell cytoplasm.

IL-23 provides a limited mechanism of resistance to acute toxoplasmosis in the absence of IL-12.

IL-23 and IL-12 are heterodimeric cytokines which share the p40 subunit, but which have unique second subunits, IL-23p19 and IL-12p35. Since p40 is required for the development of the Th1 type response necessary for resistance to Toxoplasma gondii, studies were performed to assess the role of IL-23 in resistance to this pathogen. Increased levels of IL-23 were detected in mice infected with T. gondii and in vitro stimulation of dendritic cells with this pathogen resulted in increased levels of mRNA for this cytokine. To address the role of IL-23 in resistance to T. gondii, mice lacking the p40 subunit (common to IL-12 and IL-23) and mice that lack IL-12 p35 (specific for IL-12) were infected and their responses were compared. These studies revealed that p40(-/-) mice rapidly succumbed to toxoplasmosis, while p35(-/-) mice displayed enhanced resistance though they eventually succumbed to this infection. In addition, the administration of IL-23 to p40(-/-) mice infected with T. gondii resulted in a decreased parasite burden and enhanced resistance. However, the enhanced resistance of p35(-/-) mice or p40(-/-) mice treated with IL-23 was not associated with increased production of IFN-gamma. When IL-23p19(-/-) mice were infected with T. gondii these mice developed normal T cell responses and controlled parasite replication to the same extent as wild-type mice. Together, these studies indicate that IL-12, not IL-23, plays a dominant role in resistance to toxoplasmosis but, in the absence of IL-12, IL-23 can provide a limited mechanism of resistance to this infection.

Aberrant development of Plasmodium falciparum in hemoglobin CC red cells: implications for the malaria protective effect of the homozygous state.

Although selection of hemoglobin C (HbC) by malaria has been speculated for decades, only recently have epidemiologic studies provided support for HbC protection against malaria in West Africa. A reduced risk of malaria associated with the homozygous CC state has been attributed to the inability of CC cells to support parasite multiplication in vitro. However, there have been conflicting data and conclusions regarding the ability of CC red cells to support parasite replication. Reports that parasites cannot multiply in CC cells in vitro contrast with detection of substantial parasite densities in CC patients with malaria. We have therefore investigated Plasmodium falciparum growth in CC cells in vitro. Our data show that the multiplication rate of several P falciparum lines is measurable in CC cells, but lower than that in AA (HbA-normal) cells. A high proportion of ring forms and trophozoites disintegrates within a subset of CC cells, an observation that accounts for the overall lower replication rate. In addition, knobs present on the surface of infected CC cells are fewer in number and morphologically aberrant when compared with those on AA cells. Events in malaria pathogenesis that involve remodeling of the erythrocyte surface and the display of parasite antigens may be affected by these knob abnormalities. Our data suggest that only a subset of CC cells supports normal parasite replication and that components of malaria protection associated with the CC state may affect the parasite's replication capacity and involve aberrant knob formation on CC cells.

Sheep as a new experimental host for Babesia divergens.

Babesia divergens was cultivated in sheep erythrocytes in RPMI 1640 supplemented with 10% Fetal Calf Serum (FCS) or sheep serum. In vitro cultures in sheep red blood cells were initiated with human erythrocytes infected in vitro with B. divergens Rouen 1987 or with gerbil blood infected with several isolates from bovine origin. After the first subcultures on sheep erythrocytes, a ten-fold multiplication of the parasites was obtained within 48 h. Erythrocytes from three splenectomized sheep were infected in vitro with B. divergens; when parasitaemia reached 10%, the animals were inoculated with homologous parasitized erythrocytes. All sheep expressed hyperthermia with a peak between the 6th and the 9th day post-infection (p-i) and a transitory parasitaemia 10 days p-i. In vitro primary cultures were performed on two of these sheep, demonstrating the parasite persistence at very low parasitaemia in the infected animals. Splenectomized sheep can be used as a new model for B. divergens chronic infection.

Cultivation of clinically significant hemoflagellates.

The hemoflagellates, Trypanosoma spp. and Leishmania spp., are causal agents of a number of parasitic diseases having a major impact on humans and domestic animals over vast areas of the globe. Among the diseases are some of the most pernicious and deadly of human afflictions: African sleeping sickness, Chagas' disease, kala-azar, and Oriental sore. The organisms have complex, pleomorphic life cycles typically involving a vertebrate and an invertebrate host, the latter serving as a vector. In the vertebrate host, they are primarily blood and tissue parasites. In their transition from one host to another, the hemoflagellates undergo morphological, physiological, and biochemical changes that facilitate their growth and subsequent transmission. A major goal in the study of the hemoflagellates has been the cultivation in vitro of both vertebrate and invertebrate stages of the organisms. The first types of media used in their cultivation, and still useful for establishment of cultures, were undefined and contained a complex of ingredients. These gave way to semidefined formulations which included tissue culture media as a base and, as a next step, addition of tissue culture cells as a feeder layer to promote parasite growth. More recently developed media are completely defined, having replaced the feeder cells with various supplements. Serum, a sometimes-variable component of the media, can be replaced by various serum substitutes. This review focuses on the hemoflagellates that infect humans, describing stages in the development of media leading to the fully defined formulations that are now available for the cultivation of many of these organisms.

In vitro cultivation of cryptosporidium species.

The in vitro cultivation of protozoan parasites of the genus Cryptosporidium has advanced significantly in recent years. These obligate, intracellular parasites colonize the epithelium of the digestive and respiratory tracts, are often difficult to obtain in significant numbers, produce durable oocysts that defy conventional chemical disinfection methods, and are persistently infectious when stored at refrigerated temperatures (4 to 8 degrees C). While continuous culture and efficient life cycle completion (oocyst production) have not yet been achieved in vitro, routine methods for parasite preparation and cell culture infection and assays for parasite life cycle development have been established. Parasite yields may be limited, but in vitro growth is sufficient to support a variety of research studies, including assessing potential drug therapies, evaluating oocyst disinfection methods, and characterizing life cycle stage development and differentiation.

In vitro cultivation of microsporidia of clinical importance.

Although attempts to develop methods for the in vitro cultivation of microsporidia began as early as 1937, the interest in the culture of these organisms was confined mostly to microsporidia that infect insects. The successful cultivation in 1969 of Encephalitozoon cuniculi, a microsporidium of mammalian origin, and the subsequent identification of these organisms as agents of human disease heightened interest in the cultivation of microsporidia. I describe the methodology as well as the cell lines, the culture media, and culture conditions used in the in vitro culture of microsporidia such as Brachiola (Nosema) algerae, Encephalitozoon cuniculi, E. hellem, E. intestinalis, Enterocytozoon bieneusi, Trachipleistophora hominis, and Vittaforma corneae that cause human disease.

Cell membrane labeling of Eimeria tenella sporozoites with the fluorescent dye PKH-67 GL for tracking parasite-host interactions.

The fluorescent cell linker dye PKH-67 GL was used as a vital stain for sporozoites of Eimeria tenella for tests on viability, invasion of cultured primary chick kidney cells, flow cytometric analysis and fluorescence microscopy. The effect of PKH-67 GL on sporozoites was tested at a range of concentrations of dye and sporozoites. In flow cytometric analysis, 0.5-40x10(-6) M of PKH-67 GL labeled sporozoites to some degree, with the percentage of labeled sporozoites increasing with higher dye concentrations. The optimum concentration was 2x10(-6) M, allowing easy observation by fluorescence microscopy. Morphological changes in the sporozoite at concentrations greater than 5x10(-6) M were accompanied by loss of viability according to a propidium iodide inclusion assay. Sporozoite penetration of primary chick kidney cells was unaffected by the optimal level of 2x10(-6) M, allowing observation of intracellular activities. Overall, the cell linker dye greatly facilitated observation of E. tenella in vitro and in flow cytometric analysis.

Eimeria adenoeides sporozoites: enhanced invasion of cultured cells previously inoculated with Eimeria acervulina sporozoites.

The effects of prior or concurrent administration of Eimeria acervulina on invasion of cultured cells by Eimeria adenoeides sporozoites and possible mechanisms of action were examined. Baby hamster kidney (BHK) cell cultures that were inoculated with E. acervulina sporozoites were significantly more permissive for invasion by E. adenoeides sporozoites than uninoculated cultures. Enhancement of invasion by E. adenoeides did not occur when the two species were inoculated into cultures concurrently, or within 30 h of each other. However, 48 and 72 h after inoculation of BHK cells with E. acervulina, invasion by E. adenoeides sporozoites was significantly greater than invasion in uninoculated cultures. At 96 h postinoculation with E. acervulina, the enhancing effect on invasion was variable. Culture media collected from E. acervulina-inoculated cultures also significantly enhanced invasion by E. adenoeides. Slight changes in proteins of E. acervulina-inoculated versus uninoculated cell cultures were detected by Western blots of biotinylated and nonbiotinylated cells. Biotinylated bands between 10 and 25 kDa increased in the inoculated cultures. In addition, when chicken anti-E. acervulina sporozoite serum was used as a probe, labeling of a 10 kDa antigen increased in the inoculated cultures.

Pathogenesis of Cryptosporidium parvum infection.

Cryptosporidium parvum can be regarded as a minimally invasive mucosal pathogen, since it invades surface epithelial cells that line the intestinal tract but does not invade deeper layers of the intestinal mucosa. Nonetheless, infection can be associated with diarrhea and marked mucosal inflammation. This article briefly reviews in vitro and in vivo models useful for studying the pathogenesis of C. parvum infection and explores the role of innate and acquired immune responses in host defense against this protozoan parasite.