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The Giardia lamblia virus (GLV) is a non-enveloped icosahedral dsRNA and endosymbiont virus that infects the zoonotic protozoan parasite Giardia duodenalis (syn. G. lamblia, G. intestinalis), which is a pathogen of mammals, including humans. Elucidating the transmission mechanism of GLV is crucial for gaining an in-depth understanding of the virulence of the virus in G. duodenalis. GLV belongs to the family Totiviridae, which infects yeast and protozoa intracellularly; however, it also transmits extracellularly, similar to the phylogenetically, distantly related toti-like viruses that infect multicellular hosts. The GLV capsid structure is extensively involved in the longstanding discussion concerning extracellular transmission in Totiviridae and toti-like viruses. Hence, this study constructed the first high-resolution comparative atomic models of two GLV strains, namely GLV-HP and GLV-CAT, which showed different intracellular localization and virulence phenotypes, using cryogenic electron microscopy single-particle analysis. The atomic models of the GLV capsids presented swapped C-terminal extensions, extra surface loops, and a lack of cap-snatching pockets, similar to those of toti-like viruses. However, their open pores and absence of the extra crown protein resemble those of other yeast and protozoan Totiviridae viruses, demonstrating the essential structures for extracellular cell-to-cell transmission. The structural comparison between GLV-HP and GLV-CAT indicates the first evidence of critical structural motifs for the transmission and virulence of GLV in G. duodenalis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The extracellular protozoan parasite Giardia duodenalis is a well-known and important causative agent of diarrhea on a global scale. Macrophage pyroptosis has been recognized as an important innate immune effector mechanism against intracellular pathogens. Yet, the effects of noninvasive Giardia infection on macrophage pyroptosis and the associated molecular triggers and regulators remain poorly defined. Here we initially observed that NLRP3 inflammasome-mediated pyroptosis was activated in Giardia -treated macrophages, and inhibition of ROS, NLRP3, or caspase-1 could block GSDMD cleavage, IL-1β, IL-18 and LDH release, and the cell viability reduction. We also confirmed that Giardia -induced NLRP3 inflammasome activation was involved in its K63 deubiquitination. Thus, six candidate deubiquitinases were screened, among which A20 was identified as an effective regulator. We then screened TLRs on macrophage membranes and found that upon stimulation TLR4 was tightly correlated to ROS enhancement, A20-mediated NLRP3 deubiquitination, and pyroptotic signaling. In addition, several Giardia -secreted proteins were predicted as trigger factors via secretome analysis, of which peptidyl-prolyl cis-trans isomerase B (PPIB) independently induced macrophage pyroptosis. This was similar to the findings from the trophozoite treatment, and also led to the TLR4-mediated activation of NLRP3 through K63 deubiquitination by A20. Collectively, the results of this study have significant implications for expanding our understanding of host defense mechanisms after infection with G. duodenalis.

Giardiasis represents a latent problem in public health due to the exceptionally pathogenic strategies of the parasite Giardia lamblia for evading the human immune system. Strains resistant to first-line drugs are also a challenge. Therefore, new antigiardial therapies are urgently needed. Here, we tested giardial arginine deiminase (GlADI) as a target against giardiasis. GlADI belongs to an essential pathway in Giardia for the synthesis of ATP, which is absent in humans. In silico docking with six thiol-reactive compounds was performed; four of which are approved drugs for humans. Recombinant GlADI was used in enzyme inhibition assays, and computational in silico predictions and spectroscopic studies were applied to follow the enzyme's structural disturbance and identify possible effective drugs. Inhibition by modification of cysteines was corroborated using Ellman's method. The efficacy of these drugs on parasite viability was assayed on Giardia trophozoites, along with the inhibition of the endogenous GlADI. The most potent drug against GlADI was assayed on Giardia encystment. The tested drugs inhibited the recombinant GlADI by modifying its cysteines and, potentially, by altering its 3D structure. Only rabeprazole and omeprazole decreased trophozoite survival by inhibiting endogenous GlADI, while rabeprazole also decreased the Giardia encystment rate. These findings demonstrate the potential of GlADI as a target against giardiasis.

Giardiasis is one of the most prevalent gastrointestinal diseases in the world. It is caused by Giardia, Giardia lamblia, a common and opportunistic zoonotic parasite. The aim of our work is to find a natural and safe alternative treatment for giardiasis, specifically, to determine if probiotic bacteria (Lactobacillus acidophilus, Bifidobacterium bifidum, and Lactobacillus helveticus) can contribute to treatment, and act as preventives. Sixty weanling albino mice, Mus musculus, were divided into control and experimental, probiotic-fed groups. We determined infection intensity, and cure and prevention rates of giardiasis through ELISA (enzyme-linked immunosorbent assay) of stool samples and histopathological comparison of intestinal tissue. In experimental groups, there was a significant reduction in infection intensity (P<0.001) on days 10, 15, and 20, while cure rate reached 87.5%. The control group showed no signs of reduced infection or cure and only the group treated with probiotics prior to infection showed significant prevention rates. In the experimental groups, intestinal changes due to giardiasis appeared 7 days post-infection. However, almost all of these changes disappeared by the 25th day. Our results suggest a beneficial and significant effect of probiotics in the prevention and treatment of giardiasis in mice., (© 2021 The Author(s).)

Background: Intestinal parasitic infections are still a serious public health problem in poor and developing countries like Ethiopia. Local epidemiological data is crucial to design and monitor prevention and control strategies. This study aimed to determine the prevalence of intestinal parasite infections and associated risk factors among patients requested for stool examination at Jimma health center, Southwest, Ethiopia., Methods: A cross-sectional study was conducted among a total of 384 patients in Jimma health center, Southwest, Ethiopia. Stool samples were collected and examined using direct wet-mount and formal-ether concentration techniques. Data were analyzed using the Chi-Square (X2) test and SPPS Version 24 and P Value<0.05 was considered for statistically significance., Results: The overall prevalence of intestinal parasite infections was found to be 79(20.6%). The infection rate was slightly higher in females 261(68%) than in males 123(32%). Eight types of intestinal parasites were identified and the highest prevalence was Giardia lamblia 25(6.5%) followed by A. lumbricoides 22(5.7%). Single parasitic infection was found among 67(17.4%) of the patients and double infection was 12(3.1%). Shoe wearing habits, Status of fingernail, Handwashing before a meal and after defecation, Source of water for bathing and drinking were significant factors(p<0.05) for intestinal parasitic infection., Conclusion: A relatively low prevalence of intestinal parasite infections was observed among patients of Jimma health center requested for the stool examination., Competing Interests: The authors have declared that no competing interests exist.

Background: The waterborne pathogens Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi and Cyclospora cayetanensis can cause intestinal diseases in humans. An understanding of their occurrence and transport in the environment is essential for accurate quantitative microbial risk assessment., Methods: A total of 238 influent samples were collected from four wastewater treatment plants (WWTPs) and 88 samples from eight sewer locations in Guangzhou, China. PCR-based tools were used to detect and genetically characterize Cryptosporidium spp., G. duodenalis and E. bieneusi. Eimeria spp. and Cyclospora spp. were also analyzed to assess the sources of Cryptosporidium spp., G. duodenalis and E. bieneusi in wastewater., Results: The overall occurrence rates in the WWTP and sewer samples were 14.3% (34/238) and 13.6% (12/88) for Cryptosporidium spp., 55.5% (132/238) and 33.0% (29/88) for G. duodenalis, 56.3% (134/238) and 26.1% (23/88) for E. bieneusi and 45.4% (108/238) and 47.7% (42/88) for Eimeria spp., respectively. Altogether, 11 Cryptosporidium species and genotypes, six G. duodenalis genotypes, 11 E. bieneusi genotypes and four C. cayetanensis were found, together with the presence of nine Eimeria species. The common occurrence of Cryptosporidium rat genotype IV, C. muris and Eimeria papillata and E. nieschulzi suggested that rodents were significant sources of the enteric pathogens detected in the wastewater samples., Conclusions: While the dominant Cryptosporidium spp. detected in the raw wastewater sampled in this study are not pathogenic to humans, the widely detected G. duodenalis assemblage A and E. bieneusi genotypes D and Type IV are well-known zoonotic pathogens. Further studies are needed to monitor the occurrence of these waterborne pathogens in WWTPs to better understand their transmission and environmental transport in China.

Giardiasis is the major water-borne diarrheal disease present worldwide caused by the common intestinal parasite, Giardia duodenalis. This work aims to investigate the effect of G. duodenalis infection pathogenicity in immunosuppressed animals through histopathological examination. A total of 45 BALB/c mice were divided into four groups; G1 (negative control), G2 (healthy animals exposed to Giardia); G3 (immunosuppressed animals exposed to Giardia), and G4 (non-exposed immunosuppressed animals). Our study revealed that G3 was the most affected group with an infection rate of 100%. The animals showed general weakness, soft stool, and high death rate with severe histopathological changes in the duodenum and mild degenerative changes in hepatic tissues. In G2, the maximal lesions in both duodenum and liver were on the 11 th day. We spotted damage in the villi, edema in the central core, and submucosa, in addition to increased cellular infiltration with inflammation in lamina propria. The presence of the parasites within the villi and the lumen was clear. Most of the hepatocytes revealed hydropic and fatty changes, also dilated congested central veins and edema were observed. G3 changes were more intense than G2 with massive Giardia trophozoites between the intestinal villi, lumen, and extensive fatty liver degeneration. Immune suppression plays a significant role in the severity of injury with the Giardia parasites in duodenum and liver cells.

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., (© 2020 John Wiley & Sons Ltd.)

Mast cells have been shown to affect the control of infections with the protozoan parasite Giardia intestinalis . Recently, we demonstrated that Giardia excretory-secretory proteins inhibited the activity of the connective tissue mast cell-specific protease chymase. To study the potential role of the chymase mouse mast cell protease (mMCP)-4 during infections with Giardia, mMCP-4 +/+ and mMCP-4 -/- littermate mice were gavage-infected with G. intestinalis trophozoites of the human assemblage B isolate GS. No significant changes in weight gain was observed in infected young (≈10 weeks old) mMCP-4 -/- and mMCP-4 +/+ littermate mice. In contrast, infections of mature adult mice (>18 weeks old) caused significant weight loss as compared to uninfected control mice. We detected a more rapid weight loss in mMCP-4 -/- mice as compared to littermate mMCP-4 +/+ mice. Submucosal mast cell and granulocyte counts in jejunum increased in the infected adult mMCP-4 -/- and mMCP-4 +/+ mice. This increase was correlated with an augmented intestinal trypsin-like and chymotrypsin-like activity, but the myeloperoxidase activity was constant. Infected mice showed a significantly lower intestinal neutrophil elastase (NE) activity, and in vitro, soluble Giardia proteins inhibited human recombinant NE. Serum levels of IL-6 were significantly increased eight and 13 days post infection (dpi), while intestinal IL-6 levels showed a trend to significant increase 8 dpi. Strikingly, the lack of mMCP-4 resulted in significantly less intestinal transcriptional upregulation of IL-6, TNF-α, IL-25, CXCL2, IL-2, IL-4, IL-5, and IL-10 in the Giardia -infected mature adult mice, suggesting that chymase may play a regulatory role in intestinal cytokine responses.

We prepared a series of 10 carbamates derivatives based on two common antiprotozoal drugs: metronidazole ( 1 - 5 ) and secnidazole ( 6 - 10 ). The compounds were tested in vitro against a set of two amitochondriate protozoa: Giardia duodenalis and Trichomonas vaginalis . Compounds 1-10 showed strong antiprotozoal activities, with potency values in the low micromolar-to-nanomolar range, being more active than their parent drugs. Metronidazole carbamate (1) was the most active of the series, with nanomolar activities against G. duodenalis (IC 50 = 460 nM) and T. vaginalis (IC 50 = 60 nM). The potency of compound 1 was 10 times greater than that of metronidazole against both parasites. None of compounds showed in vitro cytotoxicity against VERO cells tested at 100 µM. Molecular dynamics of compounds 1-10, secnidazole, and metronidazole onto the ligand binding site of pyruvate-ferredoxin oxidoreductase of T. vaginalis and the modeled -tubulin of G. duodenalis revealed putative molecular interactions with key residues in the binding site of both proteins implicated in the mode of action of the parent drugs., Competing Interests: The authors declare no conflict of interest.

Giardia duodenalis (syn. Giardia intestinalis or Giardia lamblia ) infSAects over 280 million people each year and numerous animals. G. duodenalis can be subdivided into eight assemblages with different host specificity. Unculturable assemblages have so far resisted genome sequencing efforts. In this study, we isolated single and pooled cysts of assemblages C and D from dog faeces by FACS, and sequenced them using multiple displacement amplification and Illumina paired-end sequencing. The genomes of assemblages C and D were compared with genomes of assemblages A and B from humans and assemblage E from ruminants and pigs. The genomes obtained from the pooled cysts and from the single cysts were considered complete (>99 % marker genes observed) and the allelic sequence heterozygosity (ASH) values of assemblages C and D were 0.89 and 0.74 %, respectively. These ASH values were slightly higher than for assemblage B (>0.43 %) and much higher than for assemblages A and E, which ranged from 0.002 to 0.037 %. The flavohaemoglobin and 4Fe-4S binding domain family encoding genes involved in O 2 and NO detoxification were only present in assemblages A, B and E. Cathepsin B orthologs were found in all genomes. Six clades of cathepsin B orthologs contained one gene of each genome, while in three clades not all assemblages were represented. We conclude that whole-genome sequencing from a single Giardia cyst results in complete draft genomes, making the genomes of unculturable Giardia assemblages accessible. Observed differences between the genomes of assemblages C and D on one hand and the assemblages A, B and E on the other hand are possibly associated with host specificity.

Giardia lamblia is the causal agent of giardiasis, one of the most prevalent parasitosis in the world. Even though effective pharmacotherapies against this parasite are available, the disadvantages associated with its use call for the development of new antigiardial compounds. Based on the Giardia dependence on glycolysis as a main energy source, glycolytic enzymes appear to be attractive targets with antiparasitic potential. Among these, fructose 1,6-biphosphate aldolase (GlFBPA) has been highlighted as a promising target for drug design. Current efforts are based on the design of competitive inhibitors of GlFBPA; however, in the kinetic context of metabolic pathways, competitive inhibitors seem to have low potential as therapeutic agents. In this work, we performed an experimental and in silico structure-based approach to propose a non-catalytic binding site which could be used as a hot spot for antigardial drug design. The druggability of the selected binding site was experimentally tested; the alteration of the selected region by site directed mutagenesis disturbs the catalytic properties and the stability of the enzyme. A computational automated search of binding sites supported the potential of this region as functionally relevant. A preliminary docking study was performed, in order to explore the feasibility and type of molecules to be able to accommodate in the proposed binding region. Altogether, the results validate the proposed region as a specific molecular binding site with pharmacological potential.

Background: Enterocytozoon bieneusi and Giardia duodenalis are common human and animal pathogens. Studies have increasingly shown that non-human primates (NHPs) are common hosts of these two zoonotic parasites. However, few studies have explored the genetic diversity and public health potential of these pathogens in laboratory monkeys. In this study, we examined the genetic diversity of the two pathogens in crab-eating macaques (Macaca fascicularis) in a commercial facility in Hainan, China., Results: Enterocytozoon bieneusi and G. duodenalis were detected by PCR analysis in 461/1452 (31.7%) and 469/1452 (32.3%) fecal specimens from the animals, respectively. Significantly higher detection rates of E. bieneusi were detected in males (36.5%, 258/706) than in females (26.7%, 160/599; χ 2  = 14.391, P = 0.0001), in animals with loose stools (41.4%, 151/365) than those with normal stool (28.5%, 310/1087; χ 2  = 20.83, P < 0.0001), and in animals of over 3 years of age (38.6%, 135/350) than those of 1-3 years (29.6%, 326/1,102; χ 2  = 9.90, P = 0.0016). For G. duodenalis, the detection rate in males (33.4%, 236/706) was higher than in females but not statistically significant (30.2%, 181/599; χ 2  = 1.54, P = 0.2152), in monkeys with loose stools (41.1%, 150/365) than those with normal stools (29.3%, 319/1087; χ 2  = 17.25, P < 0.0001), and in monkeys of 1-3 years of age (36.6%, 403/1102) than those over 3 years (18.9%, 66/350; χ 2  = 38.11, P < 0.0001). Nine E. bieneusi genotypes were detected in this study by DNA sequence analysis of the internal transcribed spacer of the rRNA gene, namely Type IV (236/461), Peru8 (42/461), Pongo2 (27/461), Peru11 (12/461), D (4/461) and PigEbITS7 (1/461) previously seen in NHPs as well as humans, and CM1 (119/461), CM2 (17/461) and CM3 (3/461) that had been only detected in NHPs. DNA sequence analyses of the tpi, gdh and bg loci identified all G. duodenalis specimens as having assemblage B. Altogether, eight (4 known and 4 new), seven (6 known and 1 new) and seven (4 known and 3 new) subtypes were seen at the tpi, gdh and bg loci, leading to the detection of 53 multi-locus genotypes (MLG-B-hn01 to MLG-B-hn53). Most of them were genetically related to those previously seen in common Old-World monkeys., Conclusions: Data from this study indicate a common occurrence of zoonotic genotypes of E. bieneusi and assemblage B of G. duodenalis in farmed crab-eating macaques in Hainan, China.

The protozoan Giardia duodenalis is one of the parasites of primary concern in drinking water treatment plants, due to its resistance to chlorination. Another matter of concern regarding chlorination of drinking water is the formation of disinfection by-products in the presence of precursors such as natural organic matter (NOM). In this study, the effects of ozonation (5 mg L -1 ) on G. duodenalis cysts in raw surface water from a drinking water treatment plant were evaluated, and the presence and alteration of NOM were assessed, as an indicative of the potential to prevent total organic halogen (TOX) formation during post-chlorination. Following ozone treatment, the presence of damaged cysts was observed by direct immunofluorescence assay (IFA) and confirmed by the molecular approach propidium monoazide-polymerase chain reaction (PMA-PCR). Using an animal model, analysis of the intestinal tissues revealed that 80% of the animals inoculated with ozonated water were positive for trophozoites. This study shows that analysis of intestinal fragments is imperative to accurately assess animal infection following inoculation of treated cysts. More importantly, considering the low infective dose of Giardia cysts in susceptible hosts, an ozone dosage usually applied in drinking water treatment plants did not completely inactivate G. duodenalis cysts in surface water. Nonetheless, the results suggest that competitive reactions with NOM have occurred, and the applied ozone dosage has proven useful to remove NOM reactivity, and thus prevent halogenated DBP formation during post-chlorination., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Competing Interests: The authors have declared that no competing interests exist.

Background: The transmission of Cryptosporidium spp. and Giardia duodenalis into humans varies according to species/genotypes of the pathogens. Although infections with both parasites are recorded in Egypt, few data are available on the distribution of Cryptosporidium species and G. duodenalis genotypes. The present study assessed the occurrence and genetic diversity of Cryptosporidium spp. and G. duodenalis in Egyptian children., Methods: In the present study, 585 fecal specimens were collected from children eight years old and younger in three provinces (El-Dakahlia, El-Gharbia and Damietta) during March 2015 to April 2016. PCR-RFLP analysis of the small subunit rRNA gene and sequence analysis of the 60 kDa glycoprotein gene were used to detect and subtype Cryptosporidium spp., respectively, whereas PCR and sequence analyses of the triose phosphate isomerase, glutamate dehydrogenase and β-giardin genes were used to detect and genotype Giardia duodenalis., Results: The overall infection rates of Cryptosporidium spp. and G. duodenalis were 1.4% and 11.3%, respectively. The Cryptosporidium species identified included C. hominis and C. parvum, each with three subtype families. The C. hominis subtypes were IbA6G3 (n = 2), IdA17 (n = 1), IdA24 (n = 1) and IfA14G1R5 (n = 1), while C. parvum subtypes were IIdA20G1 (n = 1), IIaA15G2R1 (n = 1), and IIcA5G3a (n = 1). The G. duodenalis identified included both assemblages A (n = 31) and B (n = 34). All G. duodenalis assemblage A belonged to the anthroponotic sub-assemblage AII, while a high genetic heterogeneity was seen within assemblage B., Conclusions: Data from this study are useful in our understanding of the genetic diversity of Cryptosporidium spp. and G. duodenalis in Egypt and the potential importance of anthroponotic transmission in the epidemiology of both pathogens.

Introduction An association of Helicobacter pylori and common protozoal parasites in patients with abdominal discomfort and chronic diarrhoea is unclear and may be pathological. Materials and methods One hundred and sixty-one patients with diarrhoea were compared to 114 age and sex matched controls. Stool samples were examined by microscopy and DNA extracted for PCR with specific primers for H. pylori and protozoal parasites Blastocystis sp., Entamoeba sp. (Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii) and Giardia duodenalis (G. duodenalis). Results There was a marked difference in the presence of parasites between patients and controls: no parasite 42/75%, one parasite 42/15%, two or more parasites 16/10%, respectively (p < 0.001). Patients with diarrhoea were more likely to be infected with Blastocystis sp (p < 0.001), E. histolytica (p = 0.027) and E moshkovskii (p = 0.003). There was no difference in the frequency of H. pylori (p = 0.528), G duodenalis (p = 0.697) or E dispar (p = 0.425). Thirty-three patients and 27 controls had H. pylori infection. Of these, 22 patients and 6 controls were infected with Blastocystis sp (p = 0.001), 6 patients and no controls were infected with E. histolytica (p = 0.02), whilst 7 patents and 9 controls were infected with E dispar (p = 0.292). Conclusion In this population, diarrhoea is linked to infection with Blastocystis sp, E. histolytica and E moshkoviskii. In H. pylori infection, diarrhoea is linked to Blastocystis sp and E. histolytica infection. These associations may be linked pathogenically.

Giardia duodenalis and Cryptosporidium spp. are common gastrointestinal protozoa in mammals. Many studies have been conducted on the distribution of G. duodenalis and Cryptosporidium spp. genotypes in sheep and cattle. However, in China, information about molecular characterization and genetic analysis of G. duodenalis and Cryptosporidium spp. in goats is limited. In this study, 342 fecal samples from adult goats were collected from 12 farms in Sichuan Province, China. The occurrence of G. duodenalis and Cryptosporidium spp. in adult goats was 14.9% (51/342) and 4.7% (16/342), respectively. All G. duodenalis were identified as assemblage E, with two novel genotypes (assemblages E17 and E18) being detected at the beta-giardin (bg) locus. Based on three loci-beta-giardin (bg), triose phosphate isomerase (tpi), and glutamate dehydrogenase (gdh)-multilocus sequence typing revealed three novel multilocus genotypes (MLGs) of assemblage E (MLG-E1, E2, E3 (sc)). Small Subunit (SSU) rRNA-based PCR identified two Cryptosporidium species, namely C. xiaoi (11/16) and C. suis (5/16). This study is not only the first to report C. suis infection in adult goats in China but is also the first to use the MLG approach to identify G. duodenalis in adult goats., Competing Interests: The authors have declared that no competing interests exist.

Objective: Efficient and easy-to-use DNA extraction and purification methods are critical in implementing PCR-based diagnosis of pathogens. In order to optimize the routine clinical laboratory diagnosis of eukaryotic enteric pathogens, we compare, via quantitative PCR cycle threshold (Ct) values, the efficiency of two DNA extraction kits: the semi-automated EZ1 ® (Qiagen) and the manual QIAamp ® DNA Stool Mini Kit (Qiagen), on six protozoa: Blastocystis spp., Cryptosporidium parvum/hominis, Cyclospora cayetanensis, Dientamoeba fragilis, Giardia intestinalis and Cystoisospora belli and one microsporidia: Enterocytozoon bieneusi., Results: Whereas EZ1 ® (Qiagen) and QIAamp ® DNA Stool Mini Kit (Qiagen) yielded similar performances for the detection of Cryptosporidium spp. and D. fragilis, significant lower Ct values (p < 0.002) pointed out a better performance of EZ1 ® on the five remaining pathogens. DNA extraction using the semi-automated EZ1 ® procedure was faster and as efficient as the manual procedure in the seven eukaryotic enteric pathogens tested. This procedure is suitable for DNA extraction from stools in both clinical laboratory diagnosis and epidemiological study settings.

Background: Giardia duodenalis and Enterocytozoon bieneusi are widespread pathogens that can infect humans and various animal species. Thus far, there are only a few reports of G. duodenalis and E. bieneusi infections in ruminant wildlife. Thus, the objective of this study was to examine the prevalence of G. duodenalis and E. bieneusi in forest musk deer in Sichuan, China, as well as identifying their genotypes., Results: In total, we collected 223 faecal samples from musk deer at the Sichuan Institute of Musk Deer Breeding in Dujiangyan (n = 80) and the Maerkang Breeding Institute (n = 143). Five (2.24%) faecal samples were positive for G. duodenalis; three belonged to assemblage E, and two belonged to assemblage A based on the sequence analysis of the β-giardin (bg) gene. One sample each was found to be positive based on the glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) gene, respectively. Thirty-eight (17.04%) faecal samples were found to be E. bieneusi-positive based on the internal transcribed spacer (ITS) sequence, and only SC03 genotype was identified, which belonged to the zoonotic group 1 according to the phylogenic analysis. The infection rates were significantly different among the different geographical areas and age groups but had no apparent association with gender or clinical symptoms., Conclusions: To our knowledge, this was the first molecular characterisation of G. duodenalis and E. bieneusi in musk deer. Identification of the zoonotic genotypes indicated a potential public health threat, and our study suggested that the forest musk deer is an important carrier of these parasites.

Objective: Intestinal parasites and H. pylori are well-known for their high prevalence worldwide. Thus, the objective of this study waste assess risk factors and co-infection of intestinal parasites and H. pylori among adult patients with upper gastrointestinal complaints. A hospital-based cross sectional study was conducted among 363 consecutive adult patients from December 10, 2015 to February 30,2016. Stool and venous blood were collected for analysis of Intestinal parasites and H. pylori infection, respectively. Data was analyzed using SPSS version 16 and logistic regression analysis was carried out to assess predictors of co-infection. A p ≤ 0.05 was considered as statistically significant., Results: Helicobacter pylori IgG and intestinal parasites were detected in 70.25-38.3% of participants, respectively while G. lamblia accounted 22.3%. G. lamblia prevalence was significantly higher among H. pylori infected participants (COR: 2.76; 95% CI: 1.46-5.23), but E. hystolytica/dispar infection didn't show significant variation (p = 0.15). H. pylori and intestinal parasites concomitant co-infection was associated with male sex (AOR: 1.61; 95% CI: 1.01-2.56), consumption of river water (AOR: 1.85; 95% CI: 1.11-3.07) and ground/spring water (AOR: 4.10; 95% CI: 1.97-8.52). Thus, besides H. pylori investigation, upper gastrointestinal symptomatic patients should be screened for G. lamblia infection and other intestinal parasites.

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., (© A. Rousseau et al., published by EDP Sciences, 2018.)

Background: Giardia intestinalis is a non-invasive protozoan parasite that causes giardiasis in humans, the most common form of parasite-induced diarrhea. Disease mechanisms are not completely defined and very few virulence factors are known., Methodology: To identify putative virulence factors and elucidate mechanistic pathways leading to disease, we have used proteomics to identify the major excretory-secretory products (ESPs) when Giardia trophozoites of WB and GS isolates (assemblages A and B, respectively) interact with intestinal epithelial cells (IECs) in vitro., Findings: The main parts of the IEC and parasite secretomes are constitutively released proteins, the majority of which are associated with metabolism but several proteins are released in response to their interaction (87 and 41 WB and GS proteins, respectively, 76 and 45 human proteins in response to the respective isolates). In parasitized IECs, the secretome profile indicated effects on the cell actin cytoskeleton and the induction of immune responses whereas that of Giardia showed anti-oxidation, proteolysis (protease-associated) and induction of encystation responses. The Giardia secretome also contained immunodominant and glycosylated proteins as well as new candidate virulence factors and assemblage-specific differences were identified. A minor part of Giardia ESPs had signal peptides (29% for both isolates) and extracellular vesicles were detected in the ESPs fractions, suggesting alternative secretory pathways. Microscopic analyses showed ESPs binding to IECs and partial internalization. Parasite ESPs reduced ERK1/2 and P38 phosphorylation and NF-κB nuclear translocation. Giardia ESPs altered gene expression in IECs, with a transcriptional profile indicating recruitment of immune cells via chemokines, disturbances in glucose homeostasis, cholesterol and lipid metabolism, cell cycle and induction of apoptosis., Conclusions: This is the first study identifying Giardia ESPs and evaluating their effects on IECs. It highlights the importance of host and parasite ESPs during interactions and reveals the intricate cellular responses that can explain disease mechanisms and attenuated inflammatory responses during giardiasis.

The protozoan parasite Giardia duodenalis is responsible for more than 280 million cases of gastrointestinal complaints ("giardiasis") every year, worldwide. Infections are acquired orally, mostly via uptake of cysts in contaminated drinking water. After transformation into the trophozoite stage, parasites start to colonize the duodenum and upper jejunum where they attach to the intestinal epithelium and replicate vegetatively. Outcome of Giardia infections vary between individuals, from self-limiting to chronic, and asymptomatic to severely symptomatic infection, with unspecific gastrointestinal complaints. One proposed mechanism for pathogenesis is the breakdown of intestinal barrier function. This has been studied by analyzing trans-epithelial electric resistances (TEER) or by indicators of epithelial permeability using labeled sugar compounds in in vitro cell culture systems, mouse models or human biopsies and epidemiological studies. Here, we discuss the results obtained mainly with epithelial cell models to highlight contradictory findings. We relate published studies to our own findings that suggest a lack of barrier compromising activities of recent G. duodenalis isolates of assemblage A, B, and E in a Caco-2 model system. We propose that this epithelial cell model be viewed as mimicking asymptomatic infection. This view will likely lead to a more informative use of the model if emphasis is shifted from aiming to identify Giardia virulence factors to defining non-parasite factors that arguably appear to be more decisive for disease.

Background & Objectives: Intestinal parasitic infections and their associated complications are a major cause of morbidity in the developing world. This retrospective study was done to assess the prevalence of intestinal parasitic infections among patients in a tertiary healthcare setting and to analyze age-, gender- and time-related trends in the prevalence of these intestinal parasites over a seven year period (2006-2012)., Methods: The presence of various intestinal parasites in a tertiary care setting over a seven year period in different age groups was determined by performing routine stool microscopy. Modified acid-fast staining was performed for stool samples collected from children less than five years of age for the detection of intestinal coccidian parasites. Statistical analysis was carried out to analyze age-related trends in relation to the prevalence of commonly detected intestinal parasites. Seasonal fluctuations in parasite prevalence were evaluated by performing harmonic regression analysis., Results: A total of 257,588 stool samples were received over the seven year period for examination. The highest percentage of intestinal parasites was in the 6-10 yr age group. Among the intestinal parasites, Giardia intestinalis had the highest prevalence across most age groups, except in those above 60 yr of age where hookworm became more prevalent. A significant decreasing trend with age was observed for G. intestinalis, whereas for hookworm and Strongyloides stercoralis, an increasing trend with age was seen. Significant linear temporal trends were observed for parasites such as G. intestinalis, Entamoeba histolytica and Ascaris lumbricoides., Interpretation & Conclusions: While G. intestinalis was more common in the younger age groups, certain soil-transmitted helminths such as hookworm and S. stercoralis showed a higher prevalence in the older populations. Significant temporal trends and seasonality were observed for some of the common intestinal parasites.

The zoonotic potential of giardiasis, as proposed by WHO since the late 70's, has been largely confirmed in this century. The genetic assemblages A and B of Giardia duodenalis are frequently isolated from human and canine hosts. Most of the assemblage A strains are not infective to adult mice, which can limit the range of studies regarding to biology of G. duodenalis, including virulence factors and the interaction with host immune system. This study aimed to determine the infectivity in mice of an assemblage A Giardia duodenalis strain (BHFC1) isolated from a dog and to classify the strain in sub-assemblages (AI, AII, AIII) through the phylogenetic analysis of beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes. In addition, the proteomic profile of soluble and insoluble protein fractions of trophozoites was analyzed by 2D-electrophoresis. Accordingly, trophozoites of BHFC1 were highly infective to Swiss mice. The phylogenetic analysis of tpi and gdh revealed that BHFC1 clustered to sub-assemblage AI. The proteomic map of soluble and insoluble protein fractions led to the identification of 187 proteins of G. duodenalis, 27 of them corresponding to hypothetical proteins. Considering both soluble and soluble fractions, the vast majority of the identified proteins (n = 82) were classified as metabolic proteins, mainly associated with carbon and lipid metabolism, including 53 proteins with catalytic activity. Some of the identified proteins correspond to antigens while others can be correlated with virulence. Besides a significant complementation to the proteomic data of G. duodenalis, these data provide an important source of information for future studies on various aspects of the biology of this parasite, such as virulence factors and host and pathogen interactions., Competing Interests: The authors have declared that no competing interests exist.

Background: Parasitic infections affect tens of millions of pregnant women worldwide, and directly or indirectly lead to a spectrum of adverse maternal and fetal/placental effects. The objective of this study was to assess the prevalence of intestinal parasite infections and associated risk factors among pregnant women attending antenatal care center in Felege Hiwot Referral Hospital, Bahir Dar city, northwest Ethiopia., Methods: A cross-sectional hospital based study was conducted from November 2013 to January 2014 among 384 pregnant women. Stool samples were examined for the presence of trophozoites, cysts, oocysts, and ova using direct, formal-ether sedimentation, and modified Ziehl-Neelsen techniques., Results: An overall prevalence of 31.5 % intestinal parasite infections was recorded. Eight different species of intestinal parasites were found: two protozoan and six helminth species. The highest prevalence was due to Giardia lamblia (13.3 %) followed by Entamoeba histolytica/dispar (7.8 %), hookworm (5.5 %), Ascaris lumbricoides (2.9 %), Schistosoma mansoni (2.9 %), Strongyloides stercoralis (1.6 %), Taenia spp. (0.8 %), and Hymenolepis nana (0.3 %)., Conclusions: A relatively high prevalence of intestinal parasite infections was observed among pregnant women. Routine stool examination and provision of health education are required for early medical intervention that would affect the pregnant mothers and their foetuses.

Giardia duodenalis is responsible for the majority of parasitic gastroenteritis in humans worldwide. Host-parasite interaction models in vitro provide insights into disease and virulence and help us to understand pathogenesis. Using HT-29 intestinal epithelial cells (IEC) as a model we have demonstrated that initial sensitisation by host secretions reduces proclivity for trophozoite attachment, while inducing virulence factors. Host soluble factors triggered up-regulation of membrane and secreted proteins, including Tenascins, Cathepsin-B precursor, cystatin, and numerous Variant-specific Surface Proteins (VSPs). By comparison, host-cell attached trophozoites up-regulated intracellular pathways for ubiquitination, reactive oxygen species (ROS) detoxification and production of pyridoxal phosphate (PLP). We reason that these results demonstrate early pathogenesis in Giardia involves two independent host-parasite interactions. Motile trophozoites respond to soluble secreted signals, which deter attachment and induce expression of virulence factors. Trophozoites attached to host cells, in contrast, respond by up-regulating intracellular pathways involved in clearance of ROS, thus anticipating the host defence response.

Giardia duodenalis is the most common cause of parasitic diarrhea worldwide and a well-established risk factor for postinfectious irritable bowel syndrome. We hypothesized that Giardia-induced disruptions in host-microbiota interactions may play a role in the pathogenesis of giardiasis and in postgiardiasis disease. Functional changes induced by Giardia in commensal bacteria and the resulting effects on Caenorhabditis elegans were determined. Although Giardia or bacteria alone did not affect worm viability, combining commensal Escherichia coli bacteria with Giardia became lethal to C. elegans. Giardia also induced killing of C. elegans with attenuated Citrobacter rodentium espF and map mutant strains, human microbiota from a healthy donor, and microbiota from inflamed colonic sites of ulcerative colitis patient. In contrast, combinations of Giardia with microbiota from noninflamed sites of the same patient allowed for worm survival. The synergistic lethal effects of Giardia and E. coli required the presence of live bacteria and were associated with the facilitation of bacterial colonization in the C. elegans intestine. Exposure to C. elegans and/or Giardia altered the expression of 172 genes in E. coli. The genes affected by Giardia included hydrogen sulfide biosynthesis (HSB) genes, and deletion of a positive regulator of HSB genes, cysB, was sufficient to kill C. elegans even in the absence of Giardia. Our findings indicate that Giardia induces functional changes in commensal bacteria, possibly making them opportunistic pathogens, and alters host-microbe homeostatic interactions. This report describes the use of a novel in vivo model to assess the toxicity of human microbiota., (Copyright © 2015 the American Physiological Society.)

Intestinal parasites of dogs represent a serious threat to human health due to their zoonotic potential. Thus, metropolitan areas presenting high concentrations of pets and urban fecal contamination on public areas are at sanitary risk. Major aim of this survey was to determine prevalence of zoonotic parasites in dog fecal samples collected from public soil of Milan (north-western Italy). Differences in parasites prevalence distribution were explored by a geographical information system- (GIS-) based approach, and risk factors (human density, sizes of green parks, and dog areas) were considered. The metropolitan area was divided into 157 rectangular subareas and sampling was performed following a 1-kilometer straight transect. A total of 463 fecal samples were analyzed using centrifugation-flotation technique and ELISA to detect Giardia and Cryptosporidium coproantigens. A widespread fecal contamination of soil was highlighted, being fecal samples found in 86.8% of the subareas considered. The overall prevalence of intestinal parasites was 16.63%. Zoonotic parasites were found, such as Trichuris vulpis (3.67%), Toxocara canis (1.72%), Strongyloides stercoralis (0.86%), Ancylostomatidae (0.43%), and Dipylidium caninum (0.43%). Giardia duodenalis was the most prevalent zoonotic protozoa (11.06%), followed by Cryptosporidium (1.10%). Faeces from subareas characterized by broad green areas showed to be particularly prone to infection.

Giardia duodenalis is a zoonotic protozoan parasite with public health importance worldwide. While articles about animal model infectivity have been published for G. duodenalis, the studies have used diverse protocols and parameters to evaluate the infectivity of this protozoan parasite. Hence, the objectives of this study were to (1) conduct a meta-analysis of published literature for cyst shedding and diarrhea outcomes in animal models and (2) develop recommendations to help standardize experimental dose response studies. Results showed that, for the outcome of cyst shedding in faeces, the covariates of infective stage (cyst versus trophozoite), Giardia dose, and the interactions between doses and infective stage, as well as dose and species of experimental host, were all significant (P value ≤ 0.05). This study suggests inoculation of the experimental host with cysts rather than trophozoites and administration of higher doses of Giardia will most likely result in cyst shedding. Based on the results of this meta-analysis, the infective stage (cyst versus trophozoite), parasite dose, and the interactions between dose and infective stage, as well as dose and species of experimental host, should be considered when designing experimental dose response studies that will assist in the study of zoonotic neglected tropical diseases globally.

Intestinal parasites of dogs and cats are cosmopolitan pathogens with zoonotic potential for humans. Our investigation considered their diffusion in dogs and cats from northern Italy areas, specifically the metropolitan area of Milan and two micropolitan areas of neighboring provinces. It included the study of the level of awareness in pet owners of the zoonotic potential from these parasites. A total of 409 fresh fecal samples were collected from household dogs and cats for copromicroscopic analysis and detection of Giardia duodenalis coproantigens. The assemblages of Giardia were also identified. A questionnaire about intestinal parasites biology and zoonotic potential was submitted to 185 pet owners. The overall prevalence of intestinal parasites resulted higher in cats (47.37%-60.42%) and dogs (57.41%-43.02%) from micropolitan areas than that from the metropolis of Milan (dogs: P = 28.16%; cats: P = 32.58 %). The zoonotic parasites infecting pets under investigation were T. canis and T. cati, T. vulpis, Ancylostomatidae, and G. duodenalis assemblage A. Only 49.19% of pet owners showed to be aware of the risks for human health from canine and feline intestinal parasites. Parasitological results in pets and awareness determination in their owners clearly highlight how the role of veterinarians is important in indicating correct and widespread behaviors to reduce risks of infection for pets and humans in urban areas.

Giardia lamblia is an intestinal protozoan parasite required to survive in the environment in order to be transmitted to a new host. To ensure parasite survival, flagellated trophozoites colonizing the small intestine differentiate into non-motile environmentally-resistant cysts which are then shed in the environment. This cell differentiation process called encystation is characterized by significant morphological remodeling which includes secretion of large amounts of cyst wall material. Although much is known about the transcriptional regulation of encystation and the synthesis and trafficking of cyst wall material, the investigation of global changes in protein content and abundance during G. lamblia encystation is still unaddressed. In this study, we report on the quantitative analysis of the G. lamblia proteome during encystation using tandem mass spectrometry. Quantification of more than 1000 proteins revealed major changes in protein abundance in early, mid and late encystation, notably in constitutive secretory protein trafficking. Early stages of encystation were marked by a striking decrease of endoplasmic reticulum-targeted variant-specific surface proteins and significant increases in cytoskeleton regulatory components, NEK protein kinases and proteins involved in protein folding and glycolysis. This was in stark contrast to cells in the later stages of encystation which presented a surprisingly similar proteome composition to non-encysting trophozoites. Altogether these data constitute the first quantitative atlas of the Giardia proteome covering the whole process of encystation and point towards an important role for post-transcriptional control of gene expression in Giardia differentiation. Furthermore, our data provide a valuable resource for the community-based annotation effort of the G. lamblia genome, where almost 70% of all predicted gene models remains "hypothetical".

Giardiasis is the most common waterborne parasitic infection of the human intestine worldwide. The etiological agent, Giardia duodenalis (syn. G. intestinalis, G. lamblia), is a flagellated, binucleated protozoan parasite which infects a wide array of mammalian hosts. Human giardiasis is a true cosmopolitan pathogen, with highest prevalence in developing countries. Giardiasis can present with a broad range of clinical manifestations from asymptomatic, to acute or chronic diarrheal disease associated with abdominal pain and nausea. Most infections are self-limiting, although re-infection and chronic infection can occur. Recent evidence indicating that Giardia may cause chronic post-infectious gastrointestinal complications have made it a topic of intense research. The causes of the post-infectious clinical manifestations due to Giardia, even after complete elimination of the parasite, remain obscure. This review offers a state-of-the-art discussion on the long-term consequences of Giardia infections, from extra-intestinal manifestations, growth and cognitive deficiencies, to post-infectious irritable bowel syndrome. The discussion also sheds light on some of the novel mechanisms recently implicated in the production of these post-infectious manifestations.

Depletion of arginine is a recognized strategy that pathogens use to evade immune effector mechanisms. Depletion depends on microbial enzymes such as arginases, which are considered virulence factors. The effect is mostly interpreted as being a consequence of successful competition with host enzymes for the substrate. However, both arginases and arginine deiminases (ADI) have been associated with pathogen virulence. Both deplete arginine, but their reaction products differ. An ADI has been implicated in the virulence of Giardia duodenalis, an intestinal parasite that infects humans and animals, causing significant morbidity. Dendritic cells (DC) play a critical role in host defense and also in a murine G. duodenalis infection model. The functional properties of these innate immune cells depend on the milieu in which they are activated. Here, the dependence of the response of these cells on arginine was studied by using Giardia ADI and lipopolysaccharide-stimulated human monocyte-derived DC. Arginine depletion by ADI significantly increased tumor necrosis factor alpha and decreased interleukin-10 (IL-10) and IL-12p40 secretion. It also reduced the upregulation of surface CD83 and CD86 molecules, which are involved in cell-cell interactions. Arginine depletion also reduced the phosphorylation of S6 kinase in DC, suggesting the involvement of the mammalian target of rapamycin signaling pathway. The changes were due to arginine depletion and the formation of reaction products, in particular, ammonium ions. Comparison of NH(4)(+) and urea revealed distinct immunomodulatory activities of these products of deiminases and arginases, respectively. The data suggest that a better understanding of the role of arginine-depleting pathogen enzymes for immune evasion will have to take enzyme class and reaction products into consideration.

Water-related parasitic diseases are directly dependent on water bodies for their spread or as a habitat for indispensable intermediate or final hosts. Along with socioeconomic development and improvement of sanitation, overall prevalence is declining in the China. However, the heterogeneity in economic development and the inequity of access to public services result in considerable burden due to parasitic diseases in certain areas and populations across the country. In this review, we demonstrated three aspects of ten major water-related parasitic diseases, i.e., the biology and pathogenicity, epidemiology and recent advances in research in China. General measures for diseases control and special control strategies are summarized.

In view of the interest in genotype-specific pathogenesis in Giardia duodenalis , the aim of the present study was to examine the effects of infection with different, or mixed, G. duodenalis assemblages on the integrity of human intestinal epithelia. To that end, human epithelial cells (HCT-8) were cultured and exposed to different G. duodenalis assemblages (A, B, and E) or a combination of these assemblages. Epithelial disruption and apoptosis were evaluated by fluorescent microscopy and apoptotic oligonucleosome quantification. The results indicate that infection with trophozoites disrupts epithelial tight junctions and induces varying degrees of enterocyte apoptosis, depending on the infecting assemblage. All disruptions were caspase-3 dependent and were more pronounced when caused by a non-host specific assemblage. Furthermore, infections by isolates in combination with isolates from another assemblage enhanced the epithelial disruption and apoptosis. Further studies in vitro and in vivo are required to confirm the mechanisms of enhanced pathogenicity of mixed or non-host specific (or both) G. duodenalis infections. Findings in the present study point to the potential pathogenic importance of intra-species polyparasitism in giardiasis.

Introduction: Acute diarrhea continues to be a major cause of morbidity and mortality in children from developing countries. Determination of the frequency of diarrhea in an area, along with the proportion of disease caused by specific enteric agents of different origins, is considered the first step in controlling diarrheal diseases., Methodology: From 2005 to 2007, a hospital-based surveillance was conducted in two locations in Egypt to determine the causes of acute diarrhea in children younger than 5-years seeking treatment. Five additional enteric viral and parasitic pathogens were tested using commercially-available enzyme immunoassays (EIA) to re-evaluate the prevalence of diarrheal pathogens in undiagnosed cases., Results: Adenovirus, astrovirus, norovirus and G. lamblia were detected as the sole pathogen in 2% (n=34), 3% (n=56), 9% (n=191) and 7% (n=146) of the cases, respectively. E. histolytica was never detected as the sole pathogen. The percentage of diarrheal cases with a known cause increased significantly, from 48% (n=1,006) to 74% (n=1,568) (P < 0.0001)., Conclusion: In our study, the incorporation of immunoassays yielded useful data in identifying pathogens in previously pathogen-negative diarrhea cases.

Wastewater reuse contributes to closing the nutrient recycling loop as a sustainable way of managing water resources. Bangkok has over a thousand man-made drainage and irrigation canals for such purposes. Its use for agricultural and recreational purposes has a long tradition in rural and peri-urban areas. However, the continuation of these practices is increasingly questioned since potential health risks are an issue if such practices are not appropriately managed. The microbial and chemical quality of canal water has considerably deteriorated over the last decade, mainly because of discharged, untreated domestic and industrial wastewater. It is important to understand the health risks of wastewater reuse and identify risky behaviors from the most highly exposed actors promote the safe use of wastewater. This study assessed diarrhea infection risks caused by the use of and contact with wastewater in Klong Luang municipality, a peri-urban setting in Northern Bangkok, using quantitative microbial risk assessment. Wastewater samples were collected from canals, sewers at household level, and vegetables grown in the canals for consumption. Samples were also collected from irrigation water from the agricultural fields. Two protozoa, Giardia lamblia and Entamoeba histolytica, were quantified and analyzed by real-time PCR, exposure assessment was conducted, and finally, the risk of infection due to contact with wastewater in different scenarios was calculated. The results showed that canal water and vegetables were heavily contaminated with G. lamblia and E. histolytica. Infection risk was high in tested scenarios and largely exceeded the acceptable risk given by WHO guidelines.

The protozoan parasites Giardia duodenalis, Cryptosporidium spp., and Toxoplasma gondii are pathogens that are resistant to a number of environmental factors and pose significant risks to public health worldwide. Their environmental transmission is closely governed by the physicochemical properties of their cysts (Giardia) and oocysts (Cryptosporidium and Toxoplasma), allowing their transport, retention, and survival for months in water, soil, vegetables, and mollusks, which are the main reservoirs for human infection. Importantly, the cyst/oocyst wall plays a key role in that regard by exhibiting a complex polymeric coverage that determines the charge and hydrophobic characteristics of parasites' surfaces. Interaction forces between parasites and other environmental particles may be, in a first approximation, evaluated following the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory of colloidal stability. However, due to the molecular topography and nano- to microstructure of the cyst/oocyst surface, non-DVLO hydrophobic forces together with additional steric attractive and/or repulsive forces may play a pivotal role in controlling the parasite behavior when the organism is subjected to various external conditions. Here, we review several parameters that enhance or hinder the adhesion of parasites to other particles and surfaces and address the role of fast-emerging techniques for mapping the cyst/oocyst surface, e.g., by measuring its topology and the generated interaction forces at the nano- to microscale. We discuss why characterizing these interactions could be a crucial step for managing the environmental matrices at risk of microbial pollution.

Giardiasis is a significant cause of diarrheal disease and associated morbidity in children and adults worldwide. In addition to diarrhea, it can also lead to malnutrition and cognitive deficits in children from developing countries. Giardia duodenalis is considered to be a species complex of several assemblages, of which assemblage A and B are predominantly associated with human infections. Assemblage type has been associated with risk of occurrence of symptoms and duration of illness. Hence genotyping of giardial isolates may help understand better the epidemiology and transmission ecology of the disease in a particular setting or area. In India, prevalence rates of Giardia infection in patients with diarrhea range from 0.4% to 70%, and asymptomatic cyst passage has been found to be as high as 50% in rural southern India. In this review, the global distribution of giardial assemblage, zoonotic transmission and the association of assemblage with disease have been discussed, followed by epidemiology of giardiasis in India.

In the field of infectious diseases the multifaceted amino acid arginine has reached special attention as substrate for the hosts production of the antimicrobial agent nitric oxide (NO). A variety of infectious organisms interfere with this part of the host immune response by reducing the availability of arginine. This prompted us to further investigate additional roles of arginine during pathogen infections. As a model we used the intestinal parasite Giardia intestinalis that actively consumes arginine as main energy source and secretes an arginine-consuming enzyme, arginine deiminase (ADI). Reduced intestinal epithelial cell (IEC) proliferation is a common theme during bacterial and viral intestinal infections, but it has never been connected to arginine-consumption. Our specific question was thereby, whether the arginine-consumption by Giardia leads to reduced IEC proliferation, in addition to NO reduction. In vitro cultivation of human IEC lines in arginine-free or arginine/citrulline-complemented medium, as well as in interaction with different G. intestinalis isolates, were used to study effects on host cell replication by MTT assay. IEC proliferation was further analyzed by DNA content analysis, polyamine measurements and expressional analysis of cell cycle regulatory genes. IEC proliferation was reduced upon arginine-withdrawal and also in an arginine-dependent manner upon interaction with G. intestinalis or addition of Giardia ADI. We show that arginine-withdrawal by intestinal pathogens leads to a halt in the cell cycle in IECs through reduced polyamine levels and upregulated cell cycle inhibitory genes. This is of importance with regards to intestinal tissue homeostasis that is affected through reduced cell proliferation. Thus, the slower epithelial cell turnover helps the pathogen to maintain a more stable niche for colonization. This study also shows why supplementation therapy of diarrhea patients with arginine/citrulline is helpful and that citrulline especially should gain further attention in future treatment strategies.

Gardia intestinalis, the aetiological agent of giardiasis, one of the most common intestinal diseases in both developing and developed countries, induces a loss of epithelial barrier function and functional injuries of the enterocyte by mechanisms that remain unknown. Three possible mechanisms have been proposed: (i) Giardia may directly alter the epithelial barrier after a close interaction between the trophozoite and polarized intestinal cells, (ii) intestinal functions may be altered by factors secreted by Giardia including an 'enterotoxin', proteinases and lectins, and (iii) based on mouse studies, a mechanism involving the intervention of activated T lymphocytes. We used fully differentiated cultured human intestinal Caco-2/TC7 cells forming a monolayer and expressing several polarized functions of enterocytes of small intestine to investigate the mechanisms by which G. intestinalis induces structural and functional alterations in the host intestinal epithelium. We first report that adhesion of G. intestinalis at the brush border of enterocyte-like cells involves the lipid raft membrane microdomains of the trophozoite. We report an adhesion-dependent disorganization of the apical F-actin cytoskeleton that, in turn, results in a dramatic loss of distribution of functional brush border-associated proteins, including sucrase-isomaltase (SI), dipeptidylpeptidase IV (DPP IV) and fructose transporter, GLUT5, and a decrease in sucrose enzyme activity in G. intestinalis-infected enterocyte-like cells. We observed that the G. intestinalis trophozoite promotes an adhesion-dependent decrease in transepithelial electrical resistance (TER) accompanied by a rearrangement of functional tight junction (TJ)-associated occludin, and delocalization of claudin-1. Finally, we found that whereas the occludin rearrangement induced by G. intestinalis was related to apical F-actin disorganization, the delocalization of claudin-1 was not., (© 2011 Blackwell Publishing Ltd.)

Background: Asymptomatic carriage of Giardia intestinalis is highly prevalent among children in developing countries, and evidence regarding its role as a diarrhea-causing agent in these settings is controversial. Impaired linear growth and cognition have been associated with giardiasis, presumably mediated by malabsorption of nutrients. In a prospective cohort study, we aim to compare diarrhea rates in pre-school children with and without Giardia infection. Because the study was conducted in the context of an intervention trial assessing the effects of multi-nutrients on morbidity, we also assessed how supplementation influenced the relationship between Giardia and diarrhoea rates, and to what extent Giardia modifies the intervention effect on nutritional status., Methods and Findings: Data were collected in the context of a randomized placebo-controlled efficacy trial with 2×2 factorial design assessing the effects of zinc and/or multi-micronutrients on morbidity (n=612; height-for-age z-score <-1.5 SD). Outcomes measures were episodes of diarrhea (any reported, or with ≥3 stools in the last 24 h) and fever without localizing signs, as detected with health-facility based surveillance. Giardia was detected in stool by enzyme-linked immunosorbent assay. Among children who did not receive multi-nutrients, asymptomatic Giardia infection at baseline was associated with a substantial reduction in the rate of diarrhea (HR 0.32; 0.15-0.66) and fever without localizing signs (HR 0.56; 0.36-0.87), whereas no such effect was observed among children who received multi-nutrients (p-values for interaction 0.03 for both outcomes). This interaction was independent of age, HAZ-scores and distance to the research dispensary. There was no evidence that Giardia modified the intervention effect on nutritional status., Conclusion: Although causality of the Giardia-associated reduction in morbidity cannot be established, multi-nutrient supplementation results in a loss of this protection and thus seems to influence the proliferation or virulence of Giardia or associated intestinal pathogens.

Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host.

The risk factors associated with Giardia infection, in children hospitalized in Havana, Cuba, were recently explored. Children aged ≥5 years were more likely to be positive for Giardia infection than the younger children, with an odds ratio (OR) of 3·41 [95% confidence interval (CI) = 1·36-9·69]. The risk factors found to be associated with Giardia infection in univariate analyses were rural residence (OR = 3·01; CI = 1·23-7·35), belonging to a household that did not receive water from an aqueduct (OR = 3·27; CI = 1·21-8·91), drinking unboiled water (OR = 3·64; CI = 2·14-6·26), nail biting (OR = 3·47; CI = 1·97-6·08), eating unwashed vegetables raw (OR = 4·84; CI = 2·33-10·14), and a personal (OR = 3·23; CI = 1·58-6·59) or family history (OR = 3·96; CI = 1·53-10·47) of previous parasitic infection. In multivariate analyses, however, only two (modifiable) risk factors were found to be independently and significantly associated with Giardia infection: nail biting and eating unwashed vegetables raw. It therefore seems that, at least at the individual level, giardiasis-prevention activities in Havana should be focussed on health education to improve personal hygiene and food-related practices. If appropriately managed, the surveillance of drinking water and foodstuffs, for Giardia and other parasites, might also help to reduce the hospitalization of Cuban children.

Nitric oxide (NO) is a free radical synthesized from L-arginine by different isoforms NO-synthases. NO possesses multiple and complex biological functions. NO is an important mediator of homeostasis, and changes in its generation or actions can contribute or not to pathological states. The knowledge of effects of NO has been not only important to our understanding of immune response, but also to new tools for research and treatment of various diseases. Knowing the importance of NO as inflammatory mediator in diverse infectious diseases, we decided to develop a revision that shows the participation/effect of this mediator in immune response induced against Giardia spp. Several studies already demonstrated the participation of NO with microbicidal and microbiostatic activity in giardiasis. On the other hand, some works report that Giardia spp. inhibit NO production by consuming the intermediate metabolite arginine. In fact, studies in vitro showed that G. lamblia infection of human intestinal epithelial cells had reduced NO production. This occurs due to limited offer of the crucial substrate arginine (essential aminoacid for NO production), consequently reducing NO production. Therefore, the balance between giardial arginine consumption and epithelial NO production could contribute to the variability of the duration and severity of infections by this ubiquitous parasite.

Giardia lamblia differentiates into infectious cysts to survive outside of the host. It is of interest to identify factors involved in up-regulation of cyst wall proteins (CWPs) during this differentiation. Pax proteins are important regulators of development and cell differentiation in Drosophila and vertebrates. No member of this gene family has been reported to date in yeast, plants, or protozoan parasites. We have identified a pax-like gene (pax1) encoding a putative paired domain in the G. lamblia genome. Epitope-tagged Pax1 localized to nuclei during both vegetative growth and encystation. Recombinant Pax1 specifically bound to the AT-rich initiator elements of the encystation-induced cwp1 to -3 and myb2 genes. Interestingly, overexpression of Pax1 increased cwp1 to -3 and myb2 gene expression and cyst formation. Deletion of the C-terminal paired domain or mutation of the basic amino acids of the paired domain resulted in a decrease of the transactivation function of Pax1. Our results indicate that the Pax family has been conserved during evolution, and Pax1 could up-regulate the key encystation-induced genes to regulate differentiation of the protozoan eukaryote, G. lamblia.