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.

Giardiasis is a treatable disease, caused by the flagellated protozoan parasite, Giardia duodenalis (G. duodenalis). It is one of the most common enteric parasites found globally to cause gastrointestinal disturbances, and infections may result in long-term irritable bowel syndrome-like symptoms. It is a common misconception that giardiasis is associated with foreign travel, which results in locally acquired cases in the UK being underdiagnosed. This report highlights the findings from one large Scottish Health Board, arising from a change in testing methodology, which resulted in the screening of all stools submitted for enteric investigations for G. duodenalis. Previous selection criteria were restricted to patients with a travel history to specific regions of the world, or on the basis of certain clinical details. In this report, clinical details were recorded from samples shown to be positive using two methods: an ELISA-based antigen detection assay and microscopy. Clinical details were assessed for a total of 28 laboratory-confirmed positive cases against the original selection criteria. Twenty-six cases (93%) would have been excluded from Giardia testing if the previous selection criteria had been applied. Although nine cases stated foreign travel, only two had been to regions deemed to be ‘high risk’. Therefore, those seven cases that travelled to perceived ‘low-risk’ regions would have been excluded from testing for this reason. This summary highlights the need for significant improvements to the selection criteria for Giardia testing. Laboratories should be encouraged towards the testing of all routinely submitted stools for this neglected pathogen to ensure cases that are acquired locally are properly identified and treated effectively.

The microaerophilic protozoan parasite Giardia lamblia occurs globally and causes dysentery in humans and animals. Since it is very sensitive to oxygen and reactive oxygen species, G. lamblia disposes over several enzymatic pathways to counter oxidative stress. One of the enzymes involved is thioredoxin reductase (TrxR), a central redox regulator that indirectly reduces peroxiredoxins via thioredoxin, an electron shuttle protein. Interestingly, the components of the TrxR-mediated redox system, including functional thioredoxins, have so far not been described despite their surmised importance for parasite survival. We aimed at filling this gap and attempted to identify functional thioredoxins and other interaction partners of TrxR in G. lamblia. To this end, we conducted database searches and expressed three recombinant candidate thioredoxins in Escherichia coli for ensuing enzyme assays. Further, co-immunoprecipitation experiments were conducted in order to identify further components of the thioredoxin redox network. Finally, the cellular localization of TrxR and peroxiredoxin 1 was determined by immunofluorescence microscopy. Surprisingly, our endeavours did not result in the identification of a functional thioredoxin or other credible interaction partners of TrxR. We, therefore, conclude that there is currently no evidence for a canonical thioredoxin redox network in G. lamblia.

Giardia intestinalis is the leading parasitic aetiology of human enteric infections in the United States, with an estimated 1·2 million cases occurring annually. To better understand transmission, we analysed data on all giardiasis outbreaks reported to the Centers for Disease Control and Prevention for 1971–2011. The 242 outbreaks, affecting ~41 000 persons, resulted from waterborne (74·8%), foodborne (15·7%), person-to-person (2·5%), and animal contact (1·2%) transmission. Most (74·6%) waterborne outbreaks were associated with drinking water, followed by recreational water (18·2%). Problems with water treatment, untreated groundwater, and distribution systems were identified most often during drinking water-associated outbreak investigations; problems with water treatment declined after the 1980s. Most recreational water-associated outbreaks were linked to treated swimming venues, with pools and wading pools implicated most often. Produce was implicated most often in foodborne outbreaks. Additionally, foods were most commonly prepared in a restaurant and contaminated by a food handler. Lessons learned from examining patterns in outbreaks over time can help prevent future disease. Groundwater and distribution system vulnerabilities, inadequate pool disinfection, fruit and vegetable contamination, and poor food handler hygiene are promising targets for giardiasis prevention measures.

For many years PCR- and other DNA-based methods of pathogen detection have been available in most clinical microbiology laboratories; however, until recently these tools were not routinely exploited for the diagnosis of parasitic infections. Laboratories were initially reluctant to implement PCR as incorporation of such assays within the algorithm of tools available for the most accurate diagnosis of a large variety of parasites was unclear. With regard to diagnosis of intestinal parasitic infections, the diversity of parasites that one can expect in most settings is far less than the parasitological textbooks would have you believe, hence developing a simplified diagnostic triage is feasible. Therefore the classical algorithm based on population, patient groups, use of immuno-suppressive drugs, travel history etc. is also applicable to decide when to perform and which additional techniques are to be used, if a multiplex PCR panel is used as a first-line screening diagnostic.

Samples from different water sources (n = 396) were collected during 2009 and 2011. Wastewater (2–5 l) was purified by aluminium sulphate flocculation. Surface, ground and drinking waters (400–6400 l) were collected by filtration. Cryptosporidium oocysts and Giardia cysts were further concentrated by sucrose centrifugation. (Oo)cysts were identified by IFT (immunofluorescence test), DAPI (4′,6-diamidino-2-phenylindole) staining and DICM (difference interference contrast microscopy). Out of 206 wastewater samples, 134 (65·0%) were found to be positive for Giardia cysts and 64 (31·1%) for Cryptosporidium oocysts. Parasite numbers ranged from 0 to 2436 cysts/l and 0 to 1745 oocysts/l. Eight (4·2%) surface and drinking water samples (n = 190) were found to be positive for Giardia cysts (0–56000/100 l), and 18 (9·5%) for Cryptosporidium oocysts (2400/100 l). The purpose of this study was to establish the prevalence and concentrations of Giardia lamblia and Cryptosporidium spp. by detecting (oo)cysts from water samples. This study provides substantial evidence that G. lamblia cysts and Cryptosporidium spp. oocysts are able to enter and circulate in the aquatic environment with negative implications for public health.

Giardia lamblia is one of the most prevalent parasites of mankind and is estimated to cause over 200 million infections per year. To screen chemical libraries for compounds that perturb trophozoite proliferation we adapted a conventional culture method to 384-well plates and identified numerous inhibitors. Here we used a modified assay to screen for compounds that promote trophozoite multiplication. Trophozoite growth was reduced by dilution of the culture medium and the growth period was extended to screen 2 compound libraries comprising 1500 compounds. A total of 4 agonists of trophozoite multiplication were identified. In the presence of one of these compounds, strychnine, enhanced growth was accompanied by unusual trophozoite morphology characterized by dividing trophozoites displaying more than the 2 nuclei per cell which are normally observed. The other agonists, although belonging to 2 distinct chemical groups, are known to affect isoprenylation, indicating a link between protein or lipid isoprenylation and growth in culture. Although inhibitors of isoprenylation are known to antagonize proliferation of mammalian cells, an agonistic effect of isoprenylation modulators has to our knowledge not been described previously. These observations illustrate the power of chemical genetics for identifying pathways controlling specific traits in G. lamblia.

The clinical significance of different genetic subtypes or assemblages of Giardia lamblia is uncertain. Cases of giardiasis in south-west London between 1999 and 2005 were studied, comparing molecular-typing results with clinical and epidemiological findings from routine surveillance. We identified 819 cases, of whom 389 returned surveillance questionnaires. A subset of 267 faecal samples was submitted for typing by sequencing of the triose phosphate isomerase (tpi) and ribosomal RNA genes, and/or a separate duplex PCR of the tpi gene. Typing was successful in 199 (75%) samples by at least one of the molecular methods. Assemblage A accounted for 48 (24%) samples and Assemblage B for 145 (73%); six (3%) were mixed. Both assemblages had similar seasonality, age distribution and association with travel. Clinical features were available for 59 successfully typed cases: both assemblages caused similar illness, but Assemblage A was significantly more frequently associated with fever than Assemblage B.

Parasites that often grow anaerobically in their hosts have adopted a fermentative strategy relying on the production of partially oxidized end products, including lactate, glycerol, ethanol, succinate and acetate. This review focuses on recent progress in understanding acetate production in protist parasites, such as amoebae, diplomonads, trichomonads, trypanosomatids and in the metazoan parasites helminths, as well as the succinate production pathway(s) present in some of them. We also describe the unconventional organisation of the tricarboxylic acid cycle associated with the fermentative strategy adopted by the procyclic trypanosomes, which may resemble the probable structure of the primordial TCA cycle in prokaryotes.

Infection with Giardia lamblia often causes only minor mucosal changes to the small intestine yet frank fat malabsorption may still occur. Some evidence suggests abtext-abstract pancreatic exocrine function in subjects with giardiasis although the mechanism and significance of this is unclear. Studies were conducted in vitro to determine the effect of G. lamblia trophozoites or culture filtrates from the organism on lipolysis of triglyceride by porcine pancreatic lipase. Live trophozoites significantly inhibited lipolysis. The degree of inhibition increased with longer duration of lipase exposure to trophozoites. Total amounts of enzyme inhibited were proportional to enzyme concentration, while the percentage inhibition was greatest at lowest concentration. At a lipase concentration of 1·7 i.u./ml, enzyme activity was reduced by 89·7% compared to controls after incubation for 4 h with trophozoites. The effect was abolished using killed, intact trophozoites. Culture filtrates of G. lamblia did not inhibit lipolysis. Specificity of the effect was suggested by the failure of another flagellate protozoan, Trichomonas vaginalis, to inhibit lipase. In this assay system the inhibition of lipolysis was not dependent on the bile salt concentration present. The impact of this effect in vivo remains to be determined but it may contribute to fat malabsorption in giardiasis.

Giardia lamblia localize and multiply in the small intestine and may cause acute or chronic diarrhoea with malabsorption of fat, protein and other nutrients. Abnormal pancreatic function has been documented in giardiasis and trophozoites directly inhibit pancreatic lipase activity in vitro. The aim of this study was to examine the effect of Giardia trophozoites on pancreatic trypsin, chymotrypsin and amylase activity in vitro. Axenically cultured Giardia trophozoites (Portland-1 stock) were incubated with a range of concentrations of trypsin, chymotrypsin and amylase and enzyme activity assayed over time. Tryptic activity was decreased after incubation with Giardia trophozoites. This reduction was time dependent and linear over the incubation period of 2 h. At a trypsin concentration of 18 BAEE units/ml, there was a 35.5±4% reduction in enzyme activity after 2 h compared to controls. The total amount of activity lost was proportional to the initial trypsin concentration up to 185 BAEE units/ml. At this initial concentration, the activity was reduced by 46.5±3 units/ml after 2 h. Above this concentration, little further loss of enzyme activity was seen. To investigate the nature and specificity of this effect, similar experiments were conducted using killed trophozoites and with a related protozoan. Trichomonas vaginalis. No loss of enzyme activity was evident. Media previously incubated for 2 h with trophozoites did not diminish tryptic activity. Trophozoites had no effect on chymotrypsin or amylase activities over the range of concentrations tested. Giardia trophozoites directly diminish pancreatic tryptic activity in vitro, by an active process which appears specific and not mediated via a secretory product. This mechanism may enhance parasite survival in the small intestine by allowing trophozoites to escape destruction by luminal proteases and may also contribute to malabsorption.

The intestinal protozoan parasite Giardia lamblia causes diarrhoea in humans and animals. In the present study, we used the C57BL/6 inbred mouse model to assess the impact of a nematode (Trichinella spiralis) infection on the course of a G. lamblia (clone GS/M-83-H7) infection. Acute trichinellosis coincided with transient intestinal inflammation and generated an intestinal environment that strongly promoted growth of G. lamblia trophozoites although the local anti-Giardia immunoglobulin (Ig) A production was not affected. This increased G. lamblia infection intensity correlated with intestinal mast cell infiltration, mast cell degranulation, and total IgE production. Furthermore, a G. lamblia single-infection investigated in parallel also resulted in intestinal mast cell accumulation but severe infiltration was triggered in the absence of IgE. Recently, intestinal mast cells emerging during a G. lamblia infection were reported to be involved in those immunological mechanisms that control intestinal proliferation of the parasite in mice. This anti-giardial activity was assumed to be related to the capacity of mast cells to produce IL-6. However, this previous assumption was questioned by our present immunohistological findings indicating that murine intestinal mast cells, activated during a G. lamblia infection were IL-6-negative. In the present co-infection experiments, mast cells induced during acute trichinellosis were not able to control a concurrent G. lamblia infection. This observation makes it feasible that the T. spiralis infection created an immunological and physiological environment that superimposed the anti-giardial effect of mast cells and thus favoured intestinal growth of G. lamblia trophozoites in double-infected mice. Furthermore, our findings raise the possibility that intestinal inflammation e.g. as a consequence of a ‘pre-existing’ nematode infection is a factor which contributes to increased susceptibility of a host to a G. lamblia infection. The phenomenon of a ‘pre-existing’ nematode infection prior to a G. lamblia infection is a frequent constellation in endemic areas of giardiasis and may therefore have a direct impact on the epidemiological situation of the disease.

Giardia, a common enteric protozoan parasite is a well-recognized cause of diarrhoeal illness. The detailed mechanism of diarrhoea due to this infection is not well understood. A 58 kDa enterotoxin (ESP) was purified from the excretory–secretory product of the parasite. The present study was designed to investigate the mode of action of this enterotoxin of G. lamblia in mice enterocytes. An increase in cyclic adenosine monophosphate level, as well as intracellular Ca2+ concentration, was observed in the ESP-triggered enterocytes. The levels of phospholipase Cγ1 and inositol triphosphate were found to be upregulated. The activity of protein kinase C (PKC) in the enterocytes was also enhanced following stimulation with the ESP. An increase in the level of reactive oxygen species in ESP-stimulated cells correlated well with the decline in the activity of antioxidant enzymes (superoxide dismutase and catalase). The significantly high levels of nitrite and citrulline indicated the generation of reactive nitrogen intermediates in the ESP-triggered enterocytes. Thus, ESP could induce cross-talk among the different signal transduction pathways in the enterocytes, which could together bring about a common secretory response.

Antigenic variation of the intestinal protozoan parasite Giardia lamblia is caused by an exchange of the parasite's variant surface protein (VSP) coat. Many investigations on antigenic variation were performed with G. lamblia clone GS/M-83-H7 which produces surface antigen VSP H7. To generate novel information on giardial vsp gene transcription, vsp RNA levels were assessed by quantitative reverse transcription-(RT)-PCR in both axenic VSP H7-type trophozoites and subvariants obtained after negative selection of GS/M-83-H7 trophozoites by treatment with a cytotoxic, VSP H7-specific monoclonal antibody. Our investigation was not restricted to the assessment of the sense vsp transcript levels but also included an approach aimed at the detection of complementary antisense vsp transcripts within the two trophozoite populations. We found that sense vsp H7 RNA predominated in VSP H7-type trophozoites while sense RNA from only one (vsp IVg) of 8 subvariant vsp genes totally analysed predominated in subvariant-type trophozoites. Interestingly, the two trophozoite populations exhibited a similar relative distribution regarding the vsp H7 and vsp IVg antisense RNA molecules. An analogous sense versus antisense RNA pattern was also observed when the transcripts of gene cwp 1 (encoding cyst wall protein 1) were investigated. Here, both types of RNA molecules only appeared after cwp 1 had been induced through in vitro encystation of the parasite. These findings for the first time demonstrated that giardial antisense RNA production did not occur in a constitutive manner but was directly linked to complementary sense RNA production after activation of the respective gene systems.

Giardia lamblia is a flagellated protozoan of great medical and biological importance. It is the causative agent of giardiasis, one of the most prevalent diarrheal disease both in developed and third-world countries. Morphological studies have shown that G. lamblia does not present structures such as peroxisomes, mitochondria, and a well-elaborated Golgi complex. In this review, special emphasis is given to the contribution made by various microscopic techniques to a better knowledge of the biology of the protozoan. The application of video microscopy, immunofluorescence confocal laser scanning microscopy, and several techniques associated with transmission electron microscopy (thin section, enzyme cytochemistry, freeze-fracture, deep-etching, fracture-flip) to the study of the cell surface, peripheral vesicles, endoplasmic reticulum–Golgi complex system, and of the encystation vesicles found in trophozoites and during the process of trophozoite-cyst transformation are discussed.

The mechanism by which Giardia lamblia exerts its pathogenicity is likely to be multifactorial. A 58 kDa enterotoxin was purified and characterized from the excretory–secretory product (ESP) of the parasite (Kaur et al. 2001). In the present study an attempt has been made to elucidate the mechanism of action of the ESP, a potentially important enterotoxin. A 41 kDa glycoprotein was identified in the mouse enterocyte membrane fraction with which the ESP interacted in a GM1-specific manner. The GTPase activity was reduced in enterocytes stimulated with the ESP, resulting in an increase in the level of adenylate cyclase-dependent cyclic adenosine monophosphate (cAMP). The activity of protein kinase A (PKA) in the enterocytes was also upregulated after ESP treatment. Ultimately, a significant increase in intracellular Ca2+ concentration and decrease in cytosolic Cl− level were noticed in ESP-stimulated mouse enterocytes. Thus it is possible that the enterotoxic ESP could bind to the 41 kDa glycoprotein (receptor?) on the enterocytes and activate the G-protein-mediated signal transduction pathway resulting in alteration of electrolyte transport.

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°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.

A 58 kDa excretory–secretory product (ESP) of Giardia lamblia has been characterized. The ESP was purified over 508-fold by a combination of ammonium sulphate precipitation and sequential chromatography on affinity matrix and a gel filtration column. The homogeneity of the purified protein was established by sodium dodecyl sulphate polyacrylamide gel electrophoresis (Mr, 58 kDa) and analytical isoelectrophoresis (pI 4·75). The purified protein was recognized by the pooled sera of G. lamblia-positive patients as well as an antiserum raised against crude Giardia extract, thus indicating it to be an immunodominant parasite product. The ESP was found to agglutinate rabbit erythrocytes. The haemagglutinating activity of this protein was inhibited strongly by thyroglobulin, fetuin, asialofetuin and monosialoganglioside but not by simple sugars. The purified protein was characterized immunochemically and was found to be heat stable as well as protease sensitive. Lectin-binding studies of the purified ESP and its sensitivity to periodic-acid silver staining as well as to metaperiodate treatment clearly indicated its glycoprotein nature. The major localization site of the ESP was found to be on the surface of the parasite as revealed by flow cytometric analysis. Further, this glycoprotein induced fluid accumulation in ligated rabbit ileal loops and revealed a positive skin permeability reaction in the rabbit.