Observation of Giardia sp. in the termite gut of Heterotermes tenuis.

Giardia comprises one genus with several morphologically distinct species described in mammals (including humans, marsupials, rodents), birds, and amphibians. This group of protists provokes diarrhoea diseases in humans and animals worldwide. Transmission of the parasite occurs through the faecal-oral route. Regarding the presence of Giardia in invertebrates, some works have shown that flies can transmit Giardia cysts by contact and transport between contaminated faeces and food. In this way, flies would eventually transmit this parasite. To date, Giardia's presence in the gut of other invertebrates has not been described in the literature. Here we show by first time, using scanning electron microscopy, the presence of Giardia-like trophozoites in the gut of termite Heterotermes tenuis. Two groups of Giardia were found based exclusively on the size and the flange shape of the protozoa: one presented eight flagella, a ventral disc, size, and shape very similar to Giardia intestinalis. In contrast, other cells were smaller and showed some differences in the external morphology. We cannot exclude the possibility that they correspond to the same species and that these differences result from protozoan heterogeneity.

Insights about the structure of farnesyl diphosphate synthase (FPPS) and the activity of bisphosphonates on the proliferation and ultrastructure of Leishmania and Giardia.

BACKGROUND: The enzyme farnesyl diphosphate synthase (FPPS) is positioned in the intersection of different sterol biosynthesis pathways such as those producing isoprenoids, dolichols and ergosterol. FPPS is ubiquitous in eukaryotes and is inhibited by nitrogen-containing bisphosphonates (N-BP). N-BP activity and the mechanisms of cell death as well as damage to the ultrastructure due to N-BP has not yet been investigated in Leishmania infantum and Giardia. Thus, we evaluated the effect of N-BP on cell viability and ultrastructure and then performed structural modelling and phylogenetic analysis on the FPPS enzymes of Leishmania and Giardia. METHODS: We performed multiple sequence alignment with MAFFT, phylogenetic analysis with MEGA7, and 3D structural modelling for FPPS with Modeller 9.18 and on I-Tasser server. We performed concentration curves with N-BP in Leishmania promastigotes and Giardia trophozoites to estimate the IC50via the MTS/PMS viability method. The ultrastructure was evaluated by transmission electron microscopy, and the mechanism of cell death by flow cytometry. RESULTS: The nitrogen-containing bisphosphonate risedronate had stronger anti-proliferative activity in Leishmania compared to other N-BPs with an IC50 of 13.8 µM, followed by ibandronate and alendronate with IC50 values of 85.1 µM and 112.2 µM, respectively. The effect of N-BPs was much lower on trophozoites of Giardia than Leishmania (IC50 of 311 µM for risedronate). Giardia treated with N-BP displayed concentric membranes around the nucleus and nuclear pyknosis. Leishmania had mitochondrial swelling, myelin figures, double membranes, and plasma membrane blebbing. The same population labelled with annexin-V and 7-AAD had a loss of membrane potential (TMRE), indicative of apoptosis. Multiple sequence alignments and structural alignments of FPPS proteins showed that Giardia and Leishmania FPPS display low amino acid identity but possess the conserved aspartate-rich motifs. CONCLUSIONS: Giardia and Leishmania FPPS enzymes are phylogenetically distant but display conserved protein signatures. The N-BPs effect on FPPS was more pronounced in Leishmania than Giardia. This might be due to general differences in metabolism and differences in the FPPS catalytic site.

Microtubule organelles in Giardia.

Giardia lamblia is a widespread parasitic protist with a complex MT cytoskeleton that is critical for motility, attachment, mitosis and cell division, and transitions between its two life cycle stages-the infectious cyst and flagellated trophozoite. Giardia trophozoites have both highly dynamic and highly stable MT organelles, including the ventral disc, eight flagella, the median body and the funis. The ventral disc, an elaborate MT organelle, is essential for the parasite's attachment to the intestinal villi to avoid peristalsis. Giardia's four flagellar pairs enable swimming motility and may also promote attachment. They are maintained at different equilibrium lengths and are distinguished by their long cytoplasmic regions and novel extra-axonemal structures. The functions of the median body and funis, MT organelles unique to Giardia, remain less understood. In addition to conserved MT-associated proteins, the genome is enriched in ankyrins, NEKs, and novel hypothetical proteins that also associate with the MT cytoskeleton. High-resolution ultrastructural imaging and a current inventory of more than 300 proteins associated with Giardia's MT cytoskeleton lay the groundwork for future mechanistic analyses of parasite attachment to the host, motility, cell division, and encystation/excystation. Giardia's unique MT organelles exemplify the capacity of MT polymers to generate intricate structures that are diverse in both form and function. Thus, beyond its relevance to pathogenesis, the study of Giardia's MT cytoskeleton informs basic cytoskeletal biology and cellular evolution. With the availability of new molecular genetic tools to disrupt gene function, we anticipate a new era of cytoskeletal discovery in Giardia.

High-resolution cryo-electron microscopy on macromolecular complexes and cell organelles.

Cryo-electron microscopy techniques and computational 3-D reconstruction of macromolecular assemblies are tightly linked tools in modern structural biology. This symbiosis has produced vast amounts of detailed information on the structure and function of biological macromolecules. Typically, one of two fundamentally different strategies is used depending on the specimens and their environment. A: 3-D reconstruction based on repetitive and structurally identical unit cells that allow for averaging, and B: tomographic 3-D reconstructions where tilt-series between approximately ± 60 and ± 70° at small angular increments are collected from highly complex and flexible structures that are beyond averaging procedures, at least during the first round of 3-D reconstruction. Strategies of group A are averaging-based procedures and collect large number of 2-D projections at different angles that are computationally aligned, averaged together, and back-projected in 3-D space to reach a most complete 3-D dataset with high resolution, today often down to atomic detail. Evidently, success relies on structurally repetitive particles and an aligning procedure that unambiguously determines the angular relationship of all 2-D projections with respect to each other. The alignment procedure of small particles may rely on their packing into a regular array such as a 2-D crystal, an icosahedral (viral) particle, or a helical assembly. Critically important for cryo-methods, each particle will only be exposed once to the electron beam, making these procedures optimal for highest-resolution studies where beam-induced damage is a significant concern. In contrast, tomographic 3-D reconstruction procedures (group B) do not rely on averaging, but collect an entire dataset from the very same structure of interest. Data acquisition requires collecting a large series of tilted projections at angular increments of 1-2° or less and a tilt range of ± 60° or more. Accordingly, tomographic data collection exposes its specimens to a large electron dose, which is particularly problematic for frozen-hydrated samples. Currently, cryo-electron tomography is a rapidly emerging technology, on one end driven by the newest developments of hardware such as super-stabile microscopy stages as well as the latest generation of direct electron detectors and cameras. On the other end, success also strongly depends on new software developments on all kinds of fronts such as tilt-series alignment and back-projection procedures that are all adapted to the very low-dose and therefore very noisy primary data. Here, we will review the status quo of cryo-electron microscopy and discuss the future of cellular cryo-electron tomography from data collection to data analysis, CTF-correction of tilt-series, post-tomographic sub-volume averaging, and 3-D particle classification. We will also discuss the pros and cons of plunge freezing of cellular specimens to vitrified sectioning procedures and their suitability for post-tomographic volume averaging despite multiple artifacts that may distort specimens to some degree.

Albendazole and its derivative JVG9 induce encystation on Giardia intestinalis trophozoites.

In the present study, we evaluated the effect of an albendazole (ABZ) derivative JVG9 on cultured Giardia intestinalis. To assess the JVG9 effects, we evaluated the tubulin cytoskeleton by confocal microscopy, and we found that the characteristic staining was modified. The scanning electron microscopy images revealed extremely damaged trophozoites and cyst-like cells. The confocal images revealed that this drug triggered the expression of cyst wall protein 1 and encystation. We also found that at low doses, AL triggered the encystation process too.

Programmed cell death in Giardia.

Programmed cell death (PCD) has been observed in many unicellular eukaryotes; however, in very few cases have the pathways been described. Recently the early divergent amitochondrial eukaryote Giardia has been included in this group. In this paper we investigate the processes of PCD in Giardia. We performed a bioinformatics survey of Giardia genomes to identify genes associated with PCD alongside traditional methods for studying apoptosis and autophagy. Analysis of Giardia genomes failed to highlight any genes involved in apoptotic-like PCD; however, we were able to induce apoptotic-like morphological changes in response to oxidative stress (H2O2) and drugs (metronidazole). In addition we did not detect caspase activity in induced cells. Interestingly, we did observe changes resembling autophagy when cells were starved (staining with MDC) and genome analysis revealed some key genes associated with autophagy such as TOR, ATG1 and ATG 16. In organisms such as Trichomonas vaginalis, Entamoeba histolytica and Blastocystis similar observations have been made but no genes have been identified. We propose that Giardia possess a pathway of autophagy and a form of apoptosis very different from the classical known mechanism; this may represent an early form of programmed cell death.

Wide-field lensless fluorescent microscopy using a tapered fiber-optic faceplate on a chip.

We demonstrate lensless fluorescent microscopy over a large field-of-view of ~60 mm(2) with a spatial resolution of <4 µm. In this on-chip fluorescent imaging modality, the samples are placed on a fiber-optic faceplate that is tapered such that the density of the fiber-optic waveguides on the top facet is >5 fold larger than the bottom one. Placed on this tapered faceplate, the fluorescent samples are pumped from the side through a glass hemisphere interface. After excitation of the samples, the pump light is rejected through total internal reflection that occurs at the bottom facet of the sample substrate. The fluorescent emission from the sample is then collected by the smaller end of the tapered faceplate and is delivered to an opto-electronic sensor-array to be digitally sampled. Using a compressive sampling algorithm, we decode these raw lensfree images to validate the resolution (<4 µm) of this on-chip fluorescent imaging platform using microparticles as well as labeled Giardia muris cysts. This wide-field lensfree fluorescent microscopy platform, being compact and high-throughput, might provide a valuable tool especially for cytometry, rare cell analysis (involving large area microfluidic systems) as well as for microarray imaging applications.

Proteomic analysis of Giardia: studies from the pre- and post-genomic era.

The proteome of Giardia duodenalis has been under study for the last 25 years and has lead to the discovery of valuable information on the biology and variation of the parasite. Proteomic techniques, mainly SDS-PAGE and 2D-PAGE, have been used to investigate protein variation, cellular structure and host parasite interactions. This has allowed for the identification of assemblage and host specific proteins, structural proteins, proteins released by trophozoites upon exposure to host cell monolayers and immunoreactive proteins. These data are important in understanding the pathogenesis of G. duodenalis infections, as well as highlighting potential drug and vaccine targets. There is, however, a large amount of future work needed to fully understand the proteome of this parasite.

Giardia canis: ultrastructural analysis of G. canis trophozoites transfected with full length G. canis virus cDNA transcripts.

Giardia canis virus (GCV) is a double-stranded RNA (dsRNA) virus of the family Totiviridae. In this study, the full length cDNA of the G. canis virus was constructed in pPoly2/sfinot vector and RNA was transcribed in vitro. Virus-free G. canis trophozoites were transfected with in vitro transcribed GCV RNA by electroporation. Transfected trophozoites were cultured for 12, 24, 36, 48, 60, or 72h post transfection for analysis. The ultrastructures of the transfected trophozoites were determined by transmission electron microscopy. The viral particles were detectable sporadically in the cytoplasm as early as 24h post transfection, but became evident and wide-spread 36h post transfection. The number of viral particles increased dramatically from 48 to 60h. Viral particles were released into the culture medium starting at about 60h and detectable in nuclei 72h post transfection. Severe vacuolization was seen in transfected G. canis trophozoites as early as 36h post transfection and persisted throughout the course of this study. The results of the present study indicate that in vitro transcribed GCV transcripts were capable of infecting Giardia trophozoites, apparently replicated and packaged into mature infectious viral particles which were released from the host.

Electron microscopy and cytochemistry analysis of the endocytic pathway of pathogenic protozoa.

Endocytosis is essential for eukaryotic cell survival and has been well characterized in mammal and yeast cells. Among protozoa it is also important for evading from host immune defenses and to support intense proliferation characteristic of some life cycle stages. Here we focused on the contribution of morphological and cytochemical studies to the understanding of endocytosis in Trichomonas, Giardia, Entamoeba, Plasmodium, and trypanosomatids, mainly Trypanosoma cruzi, and also Trypanosoma brucei and Leishmania.

Identification of nucleoli in the early branching protist Giardia duodenalis.

Giardia duodenalis has been described as 'anucleolated'. In this work we analysed the subcellular distribution of several nucleolar markers in Giardia nuclei using silver and immunostaining techniques for electron and confocal laser microscopy as well as expression of epitope-tagged proteins in transgenic trophozoites. We identified anteronuclear fibrogranular structures corresponding to nucleolar organising regions with recruited ribonucleoprotein complexes, rRNA and epitope-tagged fibrillarin and rRNA-pseudouridine synthase (CBF5). Recombinant fibrillarin and CBF5 were targeted to this subcompartment. This study demonstrates the presence of nucleoli in G. duodenalis and provides a model to analyse minimal requirements for nucleolar assembly and maintenance in eukaryotic cells.

The effects of saturated fatty acids on Giardia duodenalis trophozoites in vitro.

Giardia duodenalis is a protozoal, intestinal parasite that is a common aetiological agent of infectious diarrhoea in humans worldwide. Chemotherapeutic intervention presently offers a limited range of drugs and these are usually only employed after clinical diagnosis. Moreover, these drugs are ineffective against the infectious cysts, can produce unpleasant side effects, and are expensive with limited availability in developing countries. Frequent reports of drug toxicity, treatment failure and parasite drug resistance have, in some instances, also resulted in the increasing reluctance to over-prescribe synthetic anti-microbials. Alternatively, there is now mounting evidence to suggest that some of the naturally derived, medium-chain, saturated fatty acids (MCSFAs) possess anti-microbial and anti-parasitic properties. We have therefore examined the effects of four different fatty acids on G. duodenalis trophozoites in vitro. Cytotoxicity was determined using fluorescence, scanning and transmission electron microscopic techniques and standard cytotoxicity assays. Our studies have confirmed that the MCSFA, dodecanoic acid (C: 12) (common name: lauric acid), is anti-giardial, with an LD50 concentration comparable to that of metronidazole, the drug of choice in the treatment of giardiasis. Dodecanoic acid appeared to induce trophozoite death by accumulating within the parasite cytoplasm resulting in rupture of the cell membrane. This study has opened fresh avenues for development of natural drug therapy in which food supplementation may augment, or even replace, some of the standard chemotherapeutic agents presently employed in the treatment of giardiasis and possibly other infectious intestinal diseases.

Development of species-specific rDNA probes for Giardia by multiple fluorescent in situ hybridization combined with immunocytochemical identification of cyst wall antigens.

In this study, we describe the development of fluorescent oligonucleotide probes to variable regions in the small subunit of 16S rRNA in three distinct Giardia species. Sense and antisense probes (17-22 mer) to variable regions 1, 3, and 8 were labeled with digoxygenin or selected fluorochomes (FluorX, Cy3, or Cy5). Optimal results were obtained with fluorochome-labeled oligonucleotides for detection of rRNA in Giardia cysts. Specificity of fluorescent in situ hybridization (FISH) was shown using RNase digestion and high stringency to diminish the hybridization signal, and oligonucleotide probes for rRNA in Giardia lamblia, Giardia muris, and Giardia ardeae were shown to specifically stain rRNA only within cysts or trophozoites of those species. The fluorescent oligonucleotide specific for rRNA in human isolates of Giardia was positive for ten different strains. A method for simultaneous FISH detection of cysts using fluorescent antibody (genotype marker) and two oligonucleotide probes (species marker) permitted visualization of G. lamblia and G. muris cysts in the same preparation. Testing of an environmental water sample revealed the presence of FISH-positive G. lamblia cysts with a specific rDNA probe for rRNA, while negative cysts were presumed to be of animal or bird origin.

High resolution CryoFESEM of microbial surfaces.

The outer surfaces of three microorganisms, Giardia lamblia, Enterococcus faecalis, and Proteus mirabilis, were investigated by cryo-immobilization followed by sublimation of extracellular ice and cryocoating with either Pt alone or Pt plus carbon. Cryocoated samples were examined at -125 degrees C in either an in-lens field emission SEM or a below-the-lens field emission SEM. Cryocoating with Pt alone was sufficient for low magnification observation, but attempts to do high-resolution imaging resulted in radiolysis and cracking of the specimen surface. Double coating with Pt and carbon, in combination with high resolution backscatter electron detectors, enabled high-resolution imaging of the glycocalyx of bacteria, revealing a sponge-like network over the surface. High resolution examination of bacterial flagella also revealed a periodic substructure. Common artifacts included radiolysis leading to "cracking" of the surface, and insufficient deposition of Pt resulting in the absence of detectable surface topography.

Intravascular detection of Giardia trophozoites in naturally infected mice. An electron microscopic study.

During routine transmission electron microscopic (TEM) examination of mice naturally infected with Giardia muris, an intense infection with Giardia trophozoites was demonstrated within intestinal and renal tissues. Examination of randomly taken sections from these heavily infected tissues revealed marked deep affection with mixed pathology. Duodenal sections were found loaded with Giardia trophozoites in intimate contact with necrotic gut cells. Some of these trophozoites were detected within central lacteal of damaged villi and nearby blood vessels. Interestingly, and for the first time to be demonstrated, morphologically identical G. muris trophozoite was detected in a renal blood vessel. An intense cellular immune reaction was obviously demonstrated with remarkable interaction between giant macrophages and the trophozoites particulates. Involvement of deep tissues by Giardia trophozoites and their presence within vascular channels could open up questions about the possible invasive and disseminative behavior of G. muris, particularly in heavily and naturally infected hosts.

Sequential exposure and assembly of cyst wall filaments on the surface of encysting Giardia duodenalis.

The mode of appearance and assembly of cyst wall filaments on the surface of Giardia duodenalis trophozoites committed to encyst was analysed by scanning and transmission electron microscopy (SEM and TEM) and by fluorescence microscopy (FM). SEM showed a progressive appearance of fibril patches, predominantly on the anterior area of ventral and dorsal surfaces, which then spread and coalesced. By TEM, ruthenium red (RR) displayed staining in encysting cells as rodlike spots of variable diameter (3-25 nm), possibly microfibril tips with polyanionic moieties, that displayed tangential associations and random orientations over the cell membrane. In FM assays, the 1,10-phenanthroline derivative of ruthenium red (RR/oPHE) was a specific ligand for these assembling fibrils and this staining was significantly blocked by N-acetylgalactosamine (GalNac) and galactosamine (GalN). Interestingly, RR staining was lost when the cyst wall was completely assembled and thickened as observed by TEM and FM. Kinetic FM assays, in which a mAb specific for a 26 kDa Giardia cyst wall polypeptide was used concomitantly with RR/oPHE staining, showed a differential pattern for the appearance and reactivity of polypeptide and assembling GalN/GalNac-rich moieties of Giardia cyst wall.

Treatment of giardiasis.

Giardia lamblia is both the most common intestinal parasite in the United States and a frequent cause of diarrheal illness throughout the world. In spite of its recognition as an important human pathogen, there have been relatively few agents used in therapy. This paper discusses each class of drugs used in treatment, along with their mechanism of action, in vitro and clinical efficacy, and side effects and contraindications. Recommendations are made for the preferred treatment in different clinical situations. The greatest clinical experience is with the nitroimidazole drugs, i.e., metronidazole, tinidazole, and ornidazole, which are highly effective. A 5- to 7-day course of metronidazole can be expected to cure over 90% of individuals, and a single dose of tinidazole or ornidazole will cure a similar number. Quinacrine, which is no longer produced in the United States, has excellent efficacy but may be poorly tolerated, especially in children. Furazolidone is an effective alternative but must be administered four times a day for 7 to 10 days. Paromomycin may be used during early pregnancy, because it is not systematically absorbed, but it is not always effective. Patients who have resistant infection can usually be cured by a prolonged course of treatment with a combination of a nitroimidazole with quinacrine.