A Detailed Gene Expression Map of Giardia Encystation.

Giardia intestinalis is an intestinal protozoan parasite that causes diarrheal infections worldwide. A key process to sustain its chain of transmission is the formation of infectious cysts in the encystation process. We combined deep RNAseq of a broad range of encystation timepoints to produce a high-resolution gene expression map of Giardia encystation. This detailed transcriptomic map of encystation confirmed a gradual change of gene expression along the time course of encystation, showing the most significant gene expression changes during late encystation. Few genes are differentially expressed early in encystation, but the major cyst wall proteins CWP-1 and -2 are highly up-regulated already after 3.5 h encystation. Several transcription factors are sequentially up-regulated throughout the process, but many up-regulated genes at 7, 10, and 14 h post-induction of encystation have binding sites in the upstream regions for the Myb2 transcription factor, suggesting that Myb2 is a master regulator of encystation. We observed major changes in gene expression of several meiotic-related genes from 10.5 h of encystation to the cyst stage, and at 17.5 h encystation, there are changes in many different metabolic pathways and protein synthesis. Late encystation, 21 h to cysts, show extensive gene expression changes, most of all in VSP and HCMP genes, which are involved in antigenic variation, and genes involved in chromatin modifications. This high-resolution gene expression map of Giardia encystation will be an important tool in further studies of this important differentiation process.

An update on cell division of Giardia duodenalis trophozoites.

Giardia duodenalis is a flagellated protozoan that is responsible for many cases of diarrheal disease worldwide and is characterized by its great divergence from the model organisms commonly used in studies of basic cellular processes. The life cycle of Giardia involves an infectious cyst form and a proliferative and mobile trophozoite form. Each Giardia trophozoite has two nuclei and a complex microtubule cytoskeleton that consists of eight flagellar axonemes, basal bodies, the adhesive disc, the funis and the median body. Since the success of Giardia infecting other organisms depends on its ability to divide and proliferate efficiently, Giardia must coordinate its cell division to ensure the duplication and partitioning of both nuclei and the multiple cytoskeletal structures. The purpose of this review is to summarize current knowledge about cell division and its regulation in this protist.

First report of novel assemblages and mixed infections of Giardia duodenalis in human isolates from New Zealand.

Giardia duodenalis (syn. G. intestinalis and G. lamblia) is a protozoan parasite that cause disease (giardiasis) in humans and other animals. The pathogen is classified into eight assemblages, further divided into sub-assemblages, based on genetic divergence and host specificities. There are two zoonotic subtypes known as assemblages A and B, whilst assemblages from C to H are mainly found in domesticated animals, rodents and marine mammals. Here, we report for the first time the presence of assemblage E and sub-assemblage AIII in human isolates from the South Island in New Zealand. We identified a > 99% nucleotide similarity of assemblage E and sub-assemblage AIII with sequences of the gdh gene available in GenBank from individual human samples collected in Dunedin and Christchurch, respectively. We also performed a deep sequencing approach to assess intra-host assemblage variation. The sample from Dunedin showed evidence of mixed assemblage E and zoonotic sub-assemblage BIV. The report of two novel assemblages and mixed infections provides insights into the genetic diversity, epidemiology and transmission dynamics of Giardia duodenalis in New Zealand.

Interorganellar communication and membrane contact sites in protozoan parasites.

A key characteristic of eukaryotic cells is the presence of organelles with discrete boundaries and functions. Such subcellular compartmentalization into organelles necessitates platforms for communication and material exchange between each other which often involves vesicular trafficking and associated processes. Another way is via the close apposition between organellar membranes, called membrane contact sites (MCSs). Apart from lipid transfer, MCSs have been implicated to mediate in various cellular processes including ion transport, apoptosis, and organelle dynamics. In mammalian and yeast cells, contact sites have been reported between the membranes of the following: the endoplasmic reticulum (ER) and the plasma membrane (PM), ER and the Golgi apparatus, ER and endosomes (i.e., vacuoles, lysosomes), ER and lipid droplets (LD), the mitochondria and vacuoles, the nucleus and vacuoles, and the mitochondria and lipid droplets, whereas knowledge of MCSs in non-model organisms such as protozoan parasites is extremely limited. Growing evidence suggests that MCSs play more general and conserved roles in cell physiology. In this mini review, we summarize and discuss representative MCSs in divergent parasitic protozoa, and highlight the universality, diversity, and the contribution of MCSs to parasitism.

A polo-like kinase modulates cytokinesis and flagella biogenesis in Giardia lamblia.

BACKGROUND: Polo-like kinases (PLKs) are conserved serine/threonine kinases that regulate the cell cycle. To date, the role of Giardia lamblia PLK (GlPLK) in cells has not been studied. Here, we report our investigation on the function of GlPLK to provide insight into the role of this PKL in Giardia cell division, especially during cytokinesis and flagella formation. METHODS: To assess the function of GIPLK, Giardia trophozoites were treated with the PLK-specific inhibitor GW843286X (GW). Using a putative open reading frame for the PLK identified in the Giardia genomic database, we generated a transgenic Giardia expressing hemagglutinin (HA)-tagged GlPLK and used this transgenic for immunofluorescence assays (IFAs). GlPLK expression was knocked down using an anti-glplk morpholino to observe its effect on the number of nuclei number and length of flagella. Giardia cells ectopically expressing truncated GlPLKs, kinase domain + linker (GlPLK-KDL) or polo-box domains (GlPLK-PBD) were constructed for IFAs. Mutant GlPLKs at Lys51, Thr179 and Thr183 were generated by site-directed mutagenesis and then used for the kinase assay. To elucidate the role of phosphorylated GlPLK, the phosphorylation residues were mutated and expressed in Giardia trophozoites RESULTS: After incubating trophozoites with 5 µM GW, the percentage of cells with > 4 nuclei and longer caudal and anterior flagella increased. IFAs indicated that GlPLK was localized to basal bodies and flagella and was present at mitotic spindles in dividing cells. Morpholino-mediated GlPLK knockdown resulted in the same phenotypes as those observed in GW-treated cells. In contrast to Giardia expressing GlPLK-PBD, Giardia expressing GlPLK-KDL was defective in terms of GIPLK localization to mitotic spindles and had altered localization of the basal bodies in dividing cells. Kinase assays using mutant recombinant GlPLKs indicated that mutation at Lys51 or at both Thr179 and Thr183 resulted in loss of kinase activity. Giardia expressing these mutant GlPLKs also demonstrated defects in cell growth, cytokinesis and flagella formation. CONCLUSIONS: These data indicate that GlPLK plays a role in Giardia cell division, especially during cytokinesis, and that it is also involved in flagella formation.

Giardia intestinalis can interact, change its shape and internalize large particles and microorganisms.

Giardia intestinalis is a parasitic protozoan that inhabits its vertebrate hosts' upper small intestine and is the most common cause of waterborne diarrhoea worldwide. Giardia trophozoites present few organelles, and among them, they possess peripheral vesicles (PVs), which are considered an endosomal-lysosomal system. All experimental procedures carried out until now indicate that Giardia ingests macromolecules by fluid-phase and receptor-mediated endocytic pathways. Still, there is no description concerning the interaction and ingestion of large materials. Here, we tested Giardia's capacity to interact with large particles; once, in vivo, it inhabits an environment with a microbiota. We tested protozoan interaction with yeasts, bacteria, latex beads, ferritin and albumin, in different times of interaction and used several microscopy techniques (light microscopy, scanning electron microscopy and transmission electron microscopy) to follow their fate. Giardia interacted with all of the materials we tested. Projections of the plasma membrane similar to pseudopods were seen. As albumin, small markers were found in the PVs while the larger materials were not seen there. Large vacuoles containing large latex beads were detected intracellularly. Thus, we observed that: (1) Giardia interacts with large materials; (2) Giardia can display an amoeboid shape and exhibit membrane projections when in contact with microorganisms and large inorganic materials; (3) the region of the exit of the ventral flagella is very active when in contact with large materials, although all cell surface also present activity in the interactions; (4) intracellular vacuoles, which are not the PVs, present ingested large beads.

Giardia duodenalis multi-locus genotypes in dogs with different levels of synanthropism and clinical signs.

BACKGROUND: In dogs, infections with Giardia duodenalis are mainly caused by assemblages C and D, but also by the potentially zoonotic assemblages A and B. The aims of this study were to assess differences in assemblages (i) between dogs living mainly in close proximity to humans (synanthropic dogs) versus dogs living mainly among other dogs, (ii) between samples of dogs with or without loose stool, and (iii) related to the amount of cysts shedding. METHODS: One hundred eighty-nine qPCR Giardia positive fecal samples of dogs originating from four groups (household, sheltered, hunting, and dogs for which a veterinarian sent a fecal sample to a diagnostic laboratory) were used for genotyping. For this, multi-locus genotyping of beta-giardin, triose phosphate isomerase, and glutamate dehydrogenase and genotyping of SSU rDNA gene fragments were performed. Fecal consistency was scored (loose or non-loose stool), and cysts per gram of feces were determined with qPCR. RESULTS: Assemblage D was the most prevalent in all groups, followed by the other canid assemblage C. Also, mixed C/D was common. In two (synanthropic) household dogs, the potentially zoonotic assemblage AI was present. Although occurrence of assemblage AI in household dogs was not significantly different from dogs living among other dogs (sheltered and hunting dogs), it was significantly higher compared to dogs for which a sample was sent to a diagnostic laboratory. Dogs with assemblage D shed significantly more cysts than dogs with other assemblages (except for mixed C/D results) or dogs in which no assemblage could be determined. None of the assemblages was significantly associated with loose stool. CONCLUSION: Not only do dogs mainly shed the canid Giardia duodenalis assemblages D and/or C, the numbers of cysts per gram for the canid assemblage D were also higher than for the potential zoonotic assemblage AI. Based on the assemblages shed by dogs, the risk to public health posed by dogs is estimated to be low, even though the dogs that shed AI were synanthropic household dogs. Loose stool in infected dogs was not associated with any particular Giardia assemblage.

What's eating you? An update on Giardia, the microbiome and the immune response.

Giardia intestinalis has been observed in human stools since the invention of the microscope. However, it was not recognized as a pathogen until experimental infections in humans in the 1950s resulted in diarrheal illness [1]. We now know that this protozoan is capable of inducing a malabsorptive diarrhea and that the parasite is a major contributor to stunting in young children [2]. However, the majority of infections with this parasite are not accompanied by overt diarrhea and several studies indicate that it actually has a protective effect against moderate-severe diarrhea [3]. There is therefore significant interest in the mechanisms responsible for the wide variation observed in the clinical outcomes of infection with Giardia. This review will highlight recent work on the interactions among the parasite, the host microbiome and the immune response as contributing to this variation.

Extracellular vesicles released by anaerobic protozoan parasites: Current situation.

Extracellular vesicles (EVs) have emerged as a ubiquitous mechanism for transferring information between cells and organisms across all three kingdoms of life. Parasitic unicellular eukaryotes use EVs as vehicles for intercellular communication and host manipulation. Pathogenic protozoans are able to modulate the immune system of the host and establish infection by transferring a wide range of molecules contained in different types of EVs. In addition to effects on the host, EVs are able to transfer virulence factors, drug-resistance genes and differentiation factors between parasites. In this review we cover the current knowledge on EVs from anaerobic or microaerophilic extracellular protozoan parasites, including Trichomonas vaginalis, Tritrichomonas foetus, Giardia intestinalis and Entamoeba histolytica, with a focus on their potential role in the process of infection. The role of EVs in host: parasite communication adds a new level of complexity to our understanding of parasite biology, and may be a key to understand the complexity behind their mechanism of pathogenesis.

Giardia lamblia: Laboratory Maintenance, Lifecycle Induction, and Infection of Murine Models.

Giardia lamblia is a protozoan parasite that is found ubiquitously throughout the world and is a major contributor to diarrheal disease. Giardia exhibits a biphasic lifestyle existing as either a dormant cyst or a vegetative trophozoite. Infections are typically initiated through the consumption of cyst-contaminated water or food. Giardia was first axenized in the 1970s and can be readily maintained in a laboratory setting. Additionally, Giardia is one of the few protozoans that can be induced to complete its complete lifecycle using laboratory methods. In this article, we outline protocols to maintain Giardia and induce passage through its lifecycle. We also provide protocols for infecting and quantifying parasites in an animal infection model. © 2020 Wiley Periodicals LLC. Basic Protocol 1: In vitro maintenance and growth of Giardia trophozoites Basic Protocol 2: In vitro encystation of Giardia cysts Basic Protocol 3: In vivo infections using Giardia trophozoites.

Pathogenesis and post-infectious complications in giardiasis.

Giardia is an important cause of diarrhoea, and results in post-infectious and extra-intestinal complications. This chapter presents a state-of-the art of our understanding of how this parasite may cause such abnormalities, which appear to develop at least in part in Assemblage-dependent manner. Findings from prospective longitudinal cohort studies indicate that Giardia is one of the four most prevalent enteropathogens in early life, and represents a risk factor for stunting at 2 years of age. This may occur independently of diarrheal disease, in strong support of the pathophysiological significance of the intestinal abnormalities induced by this parasite. These include epithelial malabsorption and maldigestion, increased transit, mucus depletion, and disruptions of the commensal microbiota. Giardia increases epithelial permeability and facilitates the invasion of gut bacteria. Loss of intestinal barrier function is at the core of the acute and post-infectious complications associated with this infection. Recent findings demonstrate that the majority of the pathophysiological responses triggered by this parasite can be recapitulated by the effects of its membrane-bound and secreted cysteine proteases.

Zoonotic potential of Enterocytozoon bieneusi and Giardia duodenalis in horses and donkeys in northern China.

Limited data are available on infection rates and genetic identity of Enterocytozoon bieneusi and Giardia duodenalis in horses and donkeys. In this study, 865 fecal specimens were collected from donkeys (n = 540) and horses (n = 325) in three provinces and autonomous regions in northern China during 2015-2019. Enterocytozoon bieneusi was detected and genotyped by PCR and sequence analyses of the ribosomal internal transcribed spacer (ITS) and G. duodenalis was detected and genotyped by PCR and sequence analyses of the ß-giardin, glutamate dehydrogenase, and triosephosphate isomerase genes. The overall infection rates of E. bieneusi and G. duodenalis were 21.9% (118/540) and 11.5% (62/540) in donkeys, and 7.4% (24/325) and 2.8% (9/325) in horses, respectively. These differences in infection rates of E. bieneusi and G. duodenalis between donkeys and horses were significant (χ2 = 30.9, df = 1, P < 0.0001; χ2 = 20.4, df = 1, P < 0.0001, respectively). By age, the 28.9% infection rate of E. bieneusi in donkeys under 6 months was significantly higher than that in animals over 6 months (6.0%; χ2 = 35.2, df = 1, P < 0.0001). In contrast, donkeys of 6-12 months had higher infection rate (35.9%) of G. duodenalis than donkeys under 6 months (9.9%; χ2 = 22.1, df = 1, P < 0.0001) and over 12 months (8.7%; χ2 = 17.3, df = 1, P < 0.0001). In horses, animals of > 12 months had significantly higher infection rate (31.1%) of E. bieneusi than horses under 6 months (3.4%; χ2 = 29.4, df = 1, P < 0.0001) and 6-12 months (3.8%; χ2 = 26.1, df = 1, P < 0.0001). Twenty genotypes of E. bieneusi were detected, including six known ones and 14 new genotypes. Among them, nine genotypes in 45% E. bieneusi-positive specimens belonged to the zoonotic group 1. Similarly, three G. duodenalis assemblages were detected, including A (in 2 horses and 30 donkeys), B (in 6 horses and 29 donkeys), and E (in 1 horse); three donkeys had coinfections of assemblages A and B. The assemblage A isolates identified all belong to the sub-assemblage AI. These results indicate that unlike in other farm animals, there is a common occurrence of zoonotic E. bieneusi and G. duodenalis genotypes in horses and donkeys.

DNA topoisomerase IIIß promotes cyst generation by inducing cyst wall protein gene expression in Giardia lamblia.

Giardia lamblia causes waterborne diarrhoea by transmission of infective cysts. Three cyst wall proteins are highly expressed in a concerted manner during encystation of trophozoites into cysts. However, their gene regulatory mechanism is still largely unknown. DNA topoisomerases control topological homeostasis of genomic DNA during replication, transcription and chromosome segregation. They are involved in a variety of cellular processes including cell cycle, cell proliferation and differentiation, so they may be valuable drug targets. Giardia lamblia possesses a type IA DNA topoisomerase (TOP3ß) with similarity to the mammalian topoisomerase IIIß. We found that TOP3ß was upregulated during encystation and it possessed DNA-binding and cleavage activity. TOP3ß can bind to the cwp promoters in vivo using norfloxacin-mediated topoisomerase immunoprecipitation assays. We also found TOP3ß can interact with MYB2, a transcription factor involved in the coordinate expression of cwp1-3 genes during encystation. Interestingly, overexpression of TOP3ß increased expression of cwp1-3 and myb2 genes and cyst formation. Microarray analysis confirmed upregulation of cwp1-3 and myb2 genes by TOP3ß. Mutation of the catalytically important Tyr residue, deletion of C-terminal zinc ribbon domain or further deletion of partial catalytic core domain reduced the levels of cleavage activity, cwp1-3 and myb2 gene expression, and cyst formation. Interestingly, some of these mutant proteins were mis-localized to cytoplasm. Using a CRISPR/Cas9 system for targeted disruption of top3ß gene, we found a significant decrease in cwp1-3 and myb2 gene expression and cyst number. Our results suggest that TOP3ß may be functionally conserved, and involved in inducing Giardia cyst formation.

Functional Identification of a Nuclear Localization Signal of MYB2 Protein in Giardia lamblia.

MYB2 protein was identified as a transcription factor that showed encystation-induced expression in Giardia lamblia. Although nuclear import is essential for the functioning of a transcription factor, an evident nuclear localization signal (NLS) of G. lamblia MYB2 (GlMYB2) has not been defined. Based on putative GlMYB2 NLSs predicted by 2 programs, a series of plasmids expressing hemagglutinin (HA)-tagged GlMYB2 from the promoter of G. lamblia glutamate dehydrogenase were constructed and transfected into Giardia trophozoites. Immunofluorescence assays using anti-HA antibodies indicated that GlMYB2 amino acid sequence #507-#530 was required for the nuclear localization of GlMYB2, and this sequence was named as NLSGlMYB2. We further verified this finding by demonstrating the nuclear location of a protein obtained by the fusion of NLSGlMYB2 and G. lamblia glyceraldehyde 3-phosphate dehydrogenase, a non-nuclear protein. Our data on GlMYB2 will expand our understanding on NLSs functioning in G. lamblia.

Molecular Characterization of Giardia duodenalis and Enterocytozoon bieneusi Isolated from Tibetan Sheep and Tibetan Goats Under Natural Grazing Conditions in Tibet.

In the present study, fecal samples from a total of 620 Tibetan sheep and 260 Tibetan goats from six counties in Tibet were examined by nested PCR. The results showed that the overall infection rates of Giardia duodenalis and Enterocytozoon bieneusi were 0.8% (5/620) and 15% (93/620), respectively, in Tibetan sheep, and 0% (0/260) and 9.6% (25/260), respectively, in Tibetan goats. Based on sequence analysis of the SSU rRNA, tpi, bg, and gdh genes of G. duodenalis, only assemblage E was identified. Based on sequence analysis of the ribosomal internal transcriptional spacer (ITS) region of E. bieneusi, a total of 12 genotypes (three novel and nine known) were detected, and these clustered into two separate phylogenetic groups. Genotypes CHG19, EbpA, EbpC, H, PigEBITS5, and CTS3 clustered into Group 1 with high zoonotic potential, while genotypes BEB6, CHC8, CHG1, I, CTS1, and CTS2 fell within the host-specific Group 2. Ten genotypes were detected in Tibetan sheep, and two genotypes were found in Tibetan goats. The current study indicated that E. bieneusi infections are widespread among these livestock, and Tibetan goats may play an important role as a reservoir of zoonotic E. bieneusi genotypes.

Seasonal distributions and other risk factors for Giardia duodenalis and Cryptosporidium spp. infections in dogs and cats in Chiang Mai, Thailand.

The objectives of this study were to explore risk factors associated with Giardia and Cryptosporidium infections in dogs and cats in Chiang Mai, Thailand, to describe the seasonal distributions of Giardia and Cryptosporidium prevalence, and to determine the potential for zoonotic transmission through genetic characterization of isolates. Fecal samples from 301 dogs and 66 cats were collected between August 2009 and February 2010. The presence of Giardia cysts and Cryptosporidium oocysts was determined using zinc sulfate centrifugal flotation and immunofluorescent assay (IFA). Genotype/species were determined by DNA sequence analyses of PCR products from Giardia glutamate dehydrogenase (gdh), beta-giardin (bg), and triosephosphateisomerase (tpi) and Cryptosporidium heat shock protein 70KDa (hsp70) and small subunit-rRNA (SSU-rRNA) genes. Information related to specific risk factors was collected from owners of each animal using a questionnaire. The risk factor data were analyzed for associations with Giardia and Cryptosporidium infections using logistic regression. The overall estimated prevalence of Giardia and Cryptosporidium in dogs was 25.2% and 7.6%, respectively and in cats, 27.3% and 12.1%, respectively. The estimated prevalence of Giardia infection in dogs in the rainy season (31.7%) was significantly higher than in the drier, winter season (17.2%) (p < 0.01). The estimated prevalence of Cryptosporidium infection in dogs and of Giardia and Cryptosporidium infections in cats was not associated with season (p > 0.05). Multivariable analysis indicated that Giardia cysts were more likely to be detected in fecal samples of dogs that resided in high-density environments, drank untreated water, were shedding Cryptosporidium oocysts, were having acute diarrhea or a history of chronic diarrhea, and were collected in the rainy season. All 19 Giardia PCR positive samples typed as G. duodenalis canine adapted genotypes (assemblages C or D). In cats, of six Giardia PCR positive samples, five typed as dog assemblages and one typed as assemblage AI. Of ten dogs with Cryptosporidium PCR positive samples, eight typed as C. canis, one as C. parvum (a zoonotic species) and one had both C. canis and C. parvum. Of three Cryptosporidium PCR positive samples in cats, one typed as C. felis and two typed as C. parvum. The presence of zoonotic G. duodenalis assemblage AI in a cat, and C. parvum in feces of dogs and cats suggests a potential role for a reservoir for zoonotic transmission. Whether or not these presences were from exposure to other animal or human hosts or environment are needed to be confirmed.

A 5 year retrospective analysis of common intestinal parasites at Poly Health Center, Gondar, Northwest Ethiopia.

OBJECTIVE: Intestinal parasites are present throughout the world in varying degrees of prevalence due to many factors. The aim of this study was to determine the 5-year trend prevalence of intestinal prevalence among patients who had been suspected for intestinal parasite infections. A retrospective study was conducted from 2009 to 2013 at Poly Health Center Gondar, Northwest Ethiopia. Samples were examined using direct saline wet mount methods. Statistical analysis was done using SPSS version 20 software and a P-value of < 0.05 was considered statistically significant. The results were presented in tables and graphs. RESULTS: During the study period, a total of 13,329 stool samples were requested for intestinal parasite diagnose and 5510 (41.3%) laboratory-confirmed cases were reported with a fluctuating trend. Ten different parasites were reported in each year with Entamoeba histolytica/dispar (16.8%) being the predominant parasite followed by Giardia lamblia (11.4%) and Ascaris lumbricoides (6.7%). Both males (49%) and females (51%) were equally affected (P = 0.14). The intestinal parasite was reported in all age groups in the area but the highest and the lowest prevalence were reported in age groups of 20-29 years and 40-49 years, respectively (26.5% vs 6.4%) (P < 0.001).

Long-Term Culture of Giardia lamblia in Cell Culture Medium Requires Intimate Association with Viable Mammalian Cells.

Giardia lamblia is usually cultured axenically in TYI-S-33, a complex medium which does not permit survival and growth of mammalian cells. Likewise, medium commonly used to maintain and grow mammalian cells does not support healthy trophozoite survival for more than a few hours. The inability to coculture trophozoites and epithelial cells under optimal conditions limits studies of their interactions as well as interpretation of results. Trophozoites of the WB isolate but not the GS isolate were repeatedly adapted to grow stably in long-term cocultures with Caco2, Cos7, and mouse tumor rectal (RIT) cell lines using hybridoma-screened Dulbecco's modified Eagle's medium and 10% fetal calf serum. Giardia did not grow in spent cell culture medium or when separated by a permeable membrane using transwell methodology. Giardia chronically cocultured with specific cell lines became adapted (conditioned). These Giardia cocultures grew better than nonconditioned trophozoites, and the cell lines differed in their ability to support trophozoite growth in the order of RIT > Cos7 > Caco2. Trophozoites conditioned on one cell line and then grown in the presence of a heterologous cell line changed their growth rate to that seen in conditioned Giardia from the heterologous cell line. Trophozoite survival required intimate contact with cells, suggesting that trophozoites obtain an essential nutrient or growth factor from mammalian cells. This may explain why Giardia trophozoites adhere to the small intestinal epithelium during human and animal infections. This coculture system will be useful to understand the complex interactions between the host cells and parasite.

Activity of methylgerambullin from Glycosmis species (Rutaceae) against Entamoeba histolytica and Giardia duodenalis in vitro.

Entamoeba histolytica and Giardia duodenalis are widespread intestinal protozoan parasites which both spread via cysts that have to be ingested to infect a new host. Their environment, the small intestine for G. duodenalis and the colon for E. histolytica, contains only very limited amounts of oxygen, so both parasites generate energy by fermentation and substrate level phosphorylation rather than by oxidative phosphorylation. They both contain reducing agents able to reduce and activate nitroimidazole drugs such as metronidazole which is the gold standard drug to treat Entamoeba or Giardia infections. Although metronidazole works well in the majority of cases, it has a number of drawbacks. In animal models, the drug has carcinogenic activity, and concerns about a possible teratogenic activity remain. In addition, the treatment of G. duodenalis infections is hampered by emerging metronidazole resistance. Plant-derived drugs play a dominant role in human medicine, therefore we tested the activity of 14 isolated plant compounds belonging to seven different classes in vitro against both parasites. The tests were performed in a new setting in microtiter plates under anaerobic conditions. The compound with the highest activity was methylgerambullin, a sulphur-containing amide found in Glycosmis species of the family Rutaceae with an EC50 of 14.5 µM (6.08 µg/ml) after 24 h treatment for E. histolytica and 14.6 µM (6.14 µg/ml) for G. duodenalis. The compound was successfully synthesised in the laboratory which opens the door for the generation of new derivatives with higher activity.