Characterization of a new extra-axonemal structure in the Giardia intestinalis flagella.

The inner structure of the flagella of Giardia intestinalis is similar to that of other organisms, consisting of nine pairs of outer microtubules and a central pair containing radial spokes. Although the 9+2 axonemal structure is conserved, it is not clear whether subregions, including the transition zone, are present in the flagella of this parasite. Giardia axonemes originate from basal bodies and have a lengthy cytosolic portion before becoming active flagella. The region of the emergence of the flagellum is not accompanied by any membrane specialization, as seen in other protozoa. Although Giardia is an intriguing model of study, few works focused on the ultrastructural analysis of the flagella of this parasite. Here, we analyzed the externalization region of the G. intestinalis flagella using ultra-high resolution scanning microscopy (with electrons and ions), atomic force microscopy in liquid medium, freeze fracture, and electron tomography. Our data show that this region possesses a distinctive morphological feature - it extends outward and takes on a ring-like shape. When the plasma membrane is removed, a structure surrounding the axoneme becomes visible in this region. This new extra-axonemal structure is observed in all pairs of flagella of trophozoites and remains attached to the axoneme even when the interconnections between the axonemal microtubules are disrupted. High-resolution scanning electron microscopy provided insights into the arrangement of this structure, contributing to the characterization of the externalization region of the flagella of this parasite.

Nanoarchitecture of the ventral disc of Giardia intestinalis as revealed by high-resolution scanning electron microscopy and helium ion microscopy.

The parasitic protozoan Giardia intestinalis, the causative agent of giardiasis, presents a stable and elaborated cytoskeleton, which shapes and supports several intracellular structures, including the ventral disc, the median body, the funis, and four pairs of flagella. Giardia trophozoite is the motile form that inhabits the host small intestine and attaches to epithelial cells, leading to infection. The ventral disc is considered one important element of adhesion to the intestinal cells. It is adjacent to the plasma membrane in the ventral region of the cell and consists of a spiral layer of microtubules and microribbons. In this work, we studied the organization of the cytoskeleton in the ventral disc of G. intestinalis trophozoites using high-resolution scanning electron microscopy or helium ion microscopy in plasma membrane-extracted cells. Here, we show novel morphological details about the arrangement of cross-bridges in different regions of the ventral disc. Results showed that the disc is a non-uniformly organized structure that presents specific domains, such as the margin and the ventral groove region. High-resolution scanning electron microscopy allowed observation of the labeling pattern for several anti-tubulin antibodies using secondary gold particle-labeled antibodies. Labeling in the region of the emergence of the microtubules and supernumerary microtubules using an anti-acetylated tubulin antibody was observed. Ultrastructural analysis and immunogold labeling for gamma-tubulin suggest that disc microtubules originate from a region bounded by the bands of the banded collar and merge with microtubules formed at the perinuclear region. Actin-like filaments and microtubules of the disc are associated, showing an interconnection between elements of the cytoskeleton of the trophozoite.

Cryo-EM structure of the nucleosome core particle containing Giardia lamblia histones.

Giardia lamblia is a pathogenic unicellular eukaryotic parasite that causes giardiasis. Its genome encodes the canonical histones H2A, H2B, H3, and H4, which share low amino acid sequence identity with their human orthologues. We determined the structure of the G. lamblia nucleosome core particle (NCP) at 3.6 Å resolution by cryo-electron microscopy. G. lamblia histones form a characteristic NCP, in which the visible 125 base-pair region of the DNA is wrapped in a left-handed supercoil. The acidic patch on the G. lamblia octamer is deeper, due to an insertion extending the H2B α1 helix and L1 loop, and thus cannot bind the LANA acidic patch binding peptide. The DNA and histone regions near the DNA entry-exit sites could not be assigned, suggesting that these regions are asymmetrically flexible in the G. lamblia NCP. Characterization by thermal unfolding in solution revealed that both the H2A-H2B and DNA association with the G. lamblia H3-H4 were weaker than those for human H3-H4. These results demonstrate the uniformity of the histone octamer as the organizing platform for eukaryotic chromatin, but also illustrate the unrecognized capability for large scale sequence variations that enable the adaptability of histone octamer surfaces and confer internal stability.

Giardia intestinalis coiled-coil cytolinker protein 259 interacts with actin and tubulin.

Giardia intestinalis is a human parasite that causes a diarrheal disease in developing countries. G. intestinalis has a cytoskeleton (CSK) composed of microtubules and microfilaments, and the Giardia genome does not code for the canonical CSK-binding proteins described in other eukaryotic cells. To identify candidate actin and tubulin cross-linking proteins, we performed a BLAST analysis of the Giardia genome using a spectraplakins consensus sequence as a query. Based on the highest BLAST score, we selected a 259-kDa sequence designated as a cytoskeleton linker protein (CLP259). The sequence was cloned in three fragments and characterized by immunoprecipitation, confocal microscopy, and mass spectrometry (MS). CLP259 was located in the cytoplasm in the form of clusters of thick rods and colocalized with actin at numerous sites and with tubulin in the median body. Immunoprecipitation followed by mass spectrometry revealed that CLP259 interacts with structural proteins such as giardins, SALP-1, axonemal, and eight coiled-coils. The vesicular traffic proteins detected were Mu adaptin, Vacuolar ATP synthase subunit B, Bip, Sec61 alpha, NSF, AP complex subunit beta, and dynamin. These results indicate that CLP259 in trophozoites is a CSK linker protein for actin and tubulin and could act as a scaffold protein driving vesicular traffic.

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

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

Efficacy of Ageratum conyzoides extracts against Giardia duodenalis trophozoites: an experimental study.

BACKGROUND: Giardia duodenalis causes giardiasis in humans, particularly in developing countries. Despite the availability of treatments, resistance to some of the commercial anti-Giardia drugs has been reported in addition to their harmful side effects. Therefore, novel treatments for giardiasis are required. In this study, we aimed to assess the in vitro activity of crude extracts of Ageratum conyzoides against G. duodenalis trophozoites. METHODS: Plants were classified into three groups based on their flower colors: white (W), purple (P), and white-purple (W-P). Plants were separately cut into leaf (L) and flower (F) parts. Changes in internal organelle morphology of trophozoites following exposure to crude extracts were assessed using transmission electron microscopy (TEM). In subsequent experiments, efficacy of the most active essential oils from crude extracts [half maximal inhibitory concentrations (IC50) ≤ 100 µg/mL] against G. duodenalis trophozoites was tested. In vitro anti-Giardia assays using essential oils were performed in the same way as those performed using crude extracts. RESULTS: LW-P and FP extracts showed high activity (IC50 ≤ 100 µg/mL) against G. duodenalis trophozoites, with IC50 ± SD values of 45.67 ± 0.51 and 96.00 ± 0.46 µg/mL, respectively. In subsequent experiments, IC50 ± SD values of LW-P and FP essential oils were 35.00 ± 0.50 and 89.33 ± 0.41 µg/mL, respectively. TEM revealed the degeneration of flagella and ventral discs of G. duodenalis trophozoites following exposure to crude extracts. CONCLUSION: Crude LW-P and FP extracts of A. conyzoides showed the highest activity against G. duodenalis. Exposure to crude extract induced changes in the flagella and ventral discs of G. duodenalis trophozoites, which play important roles in attachment to the surface of mucosal cells. Our results suggest that the tested extracts warrant further research in terms of their efficacy and safety as giardiasis treatment.

Exosome Biogenesis in the Protozoa Parasite Giardia lamblia: A Model of Reduced Interorganellar Crosstalk.

: Extracellular vesicles (EVs) facilitate intercellular communication and are considered a promising therapeutic tool for the treatment of infectious diseases. These vesicles involve microvesicles (MVs) and exosomes and selectively transfer proteins, lipids, mRNAs, and microRNAs from one cell to another. While MVs are formed by extrusion of the plasma membrane, exosomes are a population of vesicles of endosomal origin that are stored inside the multivesicular bodies (MVBs) as intraluminal vesicles (ILVs) and are released when the MVBs fuse with the plasma membrane. Biogenesis of exosomes may be driven by the endosomal sorting complex required for transport (ESCRT) machinery or may be ESCRT independent, and it is still debated whether these are entirely separate pathways. In this manuscript, we report that the protozoan parasite, Giardia lamblia, although lacking a classical endo-lysosomal pathway, is able to produce and release exosome-like vesicles (ElV). By using a combination of biochemical and cell biology analyses, we found that the ElVs have the same size, shape, and protein and lipid composition as exosomes described for other eukaryotic cells. Moreover, we established that some endosome/lysosome peripheral vacuoles (PVs) contain ILV during the stationary phase. Our results indicate that ILV formation and ElV release depend on the ESCRT-associated AAA+-ATPase Vps4a, Rab11, and ceramide in this parasite. Interestingly, EIV biogenesis and release seems to occur in Giardia despite the fact that this parasite has lost most of the ESCRT machinery components during evolution and is unable to produce ceramide de novo. The differences in protozoa parasite EV composition, origin, and release may reveal functional and structural properties of EVs and, thus, may provide information on cell-to-cell communication and on survival mechanisms.

The peripheral vesicles gather multivesicular bodies with different behavior during the Giardia intestinalis life cycle.

Giardia intestinalis presents an intriguing endomembrane system, which includes endoplasmic reticulum and peripheral vesicles (PVs). The PVs have previously been considered to be organelles that display early and late endosomal and lysosomal properties. Some of these vesicles accumulate macromolecules ingested by the protozoan and show acid phosphatase activity. It has been previously shown that the parasite releases microvesicles, which contribute to giardiasis pathogenesis; however, the vesicles' origin and the way in which they are released by the parasite still remain unclear. In this study, we induced the parasites to encyst in vitro and analyzed these events using advanced electron microscopy techniques, including focused ion beam and electron microscopy tomography followed by three-dimensional reconstruction, in order to better understand protozoal multivesicular body (MVB) biogenesis. In addition, we performed an ultrastructural analysis of phosphatase activity during differentiation. We demonstrated that some vegetative trophozoites' PVs exhibited morphological characteristics of MVBs with a mean diameter of 50 nm, containing intraluminal vesicles (ILVs).

On the molecular and cellular effects of omeprazole to further support its effectiveness as an antigiardial drug.

Research on Giardia lamblia has accumulated large information about its molecular cell biology and infection biology. However, giardiasis is still one of the commonest parasitic diarrheal diseases affecting humans. Additionally, an alarming increase in cases refractory to conventional treatment has been reported in low prevalence settings. Consequently, efforts directed toward supporting the efficient use of alternative drugs, and the study of their molecular targets appears promising. Repurposing of proton pump inhibitors is effective in vitro against the parasite and the toxic activity is associated with the inhibition of the G. lamblia triosephosphate isomerase (GlTIM) via the formation of covalent adducts with cysteine residue at position 222. Herein, we evaluate the effectiveness of omeprazole in vitro and in situ on GlTIM mutants lacking the most superficial cysteines. We studied the influence on the glycolysis of Giardia trophozoites treated with omeprazole and characterized, for the first time, the morphological effect caused by this drug on the parasite. Our results support the effectiveness of omeprazole against GlTIM despite of the possibility to mutate the druggable amino acid targets as an adaptive response. Also, we further characterized the effect of omeprazole on trophozoites and discuss the possible mechanism involved in its antigiardial effect.

Aminoguanidines: New leads for treatment of Giardia duodenalis infection.

Giardia duodenalis is an ubiquitous parasitic pathogen that causes significant morbidity and mortality worldwide. Failures in drug therapy are commonly due to poor patient compliance as a result of the need for repeated administration, off target drug effects and increasing parasite drug resistance. In this study the in vitro efficacy and selectivity of the aminoguanidine compound robenidine and 2 structural analogues against Giardia were determined. After 5 h exposure to each compound the IC50 was as low as 0.2 µM with corresponding MLCs as low as 2.8 µM. This is in contrast to metronidazole which required 24 h to exhibit inhibitory activity. A modified adherence assay, developed for this study, demonstrated that three of the compounds inhibited in vitro adherence of the parasite. The lead compound exhibited rapid giardicidal activity (<5hr). In addition, microscopy studies demonstrated damage to the plasma membrane of trophozoites. In conclusion, a class of aminoguanidines, represented by robenidine, has shown antigiardial activity warranting further investigation.

Unequal distribution of genes and chromosomes refers to nuclear diversification in the binucleated Giardia intestinalis.

The single-celled parasite Giardia intestinalis (Diplomonadida) has two equally sized nuclei in one cell. The nuclei have been considered identical. We have previously shown that they contain different chromosomal sets and proceed through the cell cycle with some asynchrony. Here, we demonstrate by fluorescence in situ hybridization that several genes from chromosome 5 are lost in one of the two nuclei of the WBc6 Giardia line. The missing segment stretches over at least 50 kb near the 5' chromosome end. In both WB and WBc6 Giardia cell lines, chromosome 5 is trisomic in one nucleus and monosomic in the other nucleus. The described chromosomal deletion has always been observed at the monosomic chromosome in WBc6; however, the deletion was not detected in the parent line WB. The chromosomal segment was thus initially lost after biological cloning of WB, which gave rise to clone WBc6. We show that Giardia is capable of carrying out gene expression from only one nucleus. The two nuclei display a certain level of diversity, making each of them irreplaceable. The doubled karyomastigonts of diplomonads likely have separate functions both in the mastigont/flagellar organization and in chromosomal and gene content. To our knowledge, our results offer the first methodical approach to differentiating the two, so far indistinguishable nuclei.

Proximity Staining Using Enzymatic Protein Tagging in Diplomonads.

The diplomonads are a group of understudied eukaryotic flagellates whose most prominent member is the human pathogen Giardia intestinalis Methods commonly used in other eukaryotic model systems often require special optimization in diplomonads due to the highly derived character of their cell biology. We have optimized a proximity labeling protocol using pea ascorbate peroxidase (APEX) as a reporter for transmission electron microscopy (TEM) to enable the study of ultrastructural cellular details in diplomonads. Currently available TEM-compatible tags require light-induced activation (1, 2) or are inactive in many cellular compartments (3), while ascorbate peroxidase has not been shown to have those limitations. Here, we have optimized the in vivo activities of two versions of pea ascorbate peroxidase (APXW41F and APEX) using the diplomonad fish parasite Spironucleus salmonicida, a relative of G. intestinalis We exploited the well-known peroxidase substrates, Amplex UltraRed and 3,3'-diaminobenzidine (DAB), to validate the activity of the two tags and argue that APEX is the most stable version to use in Spironucleus salmonicida Next, we fused APEX to proteins with established localization to evaluate the activity of APEX in different cellular compartments of the diplomonad cell and used Amplex UltraRed as well as antibodies along with superresolution microscopy to confirm the protein-APEX localization. The ultrastructural details of protein-APEX fusions were determined by TEM, and we observed marker activity in all cellular compartments tested when using the DAB substrate. Finally, we show that the optimized conditions established for S. salmonicida can be used in the related diplomonad G. intestinalisIMPORTANCE The function of many proteins is intrinsically related to their cellular location. Novel methods for ascertainment of the ultrastructural location of proteins have been introduced in recent years, but their implementation in protists has so far not been readily realized. Here, we present an optimized proximity labeling protocol using the APEX system in the salmon pathogen Spironucleus salmonicida This protocol was also applicable to the human pathogen Giardia intestinalis Both organisms required extraneous addition of hemin to the growth medium to enable detectable peroxidase activity. Further, we saw no inherent limitation in labeling efficiency coupled to the cellular compartment, as evident with some other proximity labeling systems. We anticipate that the APEX proximity labeling system might offer a great resource to establish the ultrastructural localization of proteins across genetically tractable protists but might require organism-specific labeling conditions.

Giardia lamblia: Identification of peroxisomal-like proteins.

The protozoan parasite Giardia lamblia has traditionally been reported as lacking peroxisomes, organelles involved in fatty acid metabolism and detoxification of reactive oxygen species. We here report the finding with transmission electron microscopy of an oxidase activity in cytoplasmic vesicles of trophozoites and cysts of G. lamblia. These vesicles were positive to 3,3'-diaminobenzidine and to cerium chloride staining. In addition, using bioinformatic tools, two peroxisomal proteins were identified in the G. lamblia proteome: acyl-CoA synthetase long chain family member 4 (ACSL-4) and peroxin-4 (PEX-4). With confocal and immunoelectron microscopy using polyclonal antibodies both proteins were identified in cytoplasmic vesicles of trophozoites. Altogether, our results suggest for the first time the presence of peroxisomal-like proteins in the cytoplasm of G. lamblia.

Physiological aspects of nitro drug resistance in Giardia lamblia.

For over 50 years, metronidazole and other nitro compounds such as nitazoxanide have been used as a therapy of choice against giardiasis and more and more frequently, resistance formation has been observed. Model systems allowing studies on biochemical aspects of resistance formation to nitro drugs are, however, scarce since resistant strains are often unstable in culture. In order to fill this gap, we have generated a stable metronidazole- and nitazoxanide-resistant Giardia lamblia WBC6 clone, the strain C4. Previous studies on strain C4 and the corresponding wild-type strain WBC6 revealed marked differences in the transcriptomes of both strains. Here, we present a physiological comparison between trophozoites of both strains with respect to their ultrastructure, whole cell activities such as oxygen consumption and resazurin reduction assays, key enzyme activities, and several metabolic key parameters such as NAD(P)+/NAD(P)H and ADP/ATP ratios and FAD contents. We show that nitro compound-resistant C4 trophozoites exhibit lower nitroreductase activities, lower oxygen consumption and resazurin reduction rates, lower ornithine-carbamyl-transferase activity, reduced FAD and NADP(H) pool sizes and higher ADP/ATP ratios than wildtype trophozoites. The present results suggest that resistance formation against nitro compounds is correlated with metabolic adaptations resulting in a reduction of the activities of FAD-dependent oxidoreductases.

New advances in scanning microscopy and its application to study parasitic protozoa.

Scanning electron microscopy has been used to observe and study parasitic protozoa for at least 40 years. However, field emission electron sources, as well as improvements in lenses and detectors, brought the resolution power of scanning electron microscopes (SEM) to a new level. Parallel to the refinement of instruments, protocols for preservation of the ultrastructure, immunolabeling, exposure of cytoskeleton and inner structures of parasites and host cells were developed. This review is focused on protozoan parasites of medical and veterinary relevance, e.g., Toxoplasma gondii, Tritrichomonas foetus, Giardia intestinalis, and Trypanosoma cruzi, compilating the main achievements in describing the fine ultrastructure of their surface, cytoskeleton and interaction with host cells. Two new resources, namely, Helium Ion Microscopy (HIM) and Slice and View, using either Focused Ion Beam (FIB) abrasion or Microtome Serial Sectioning (MSS) within the microscope chamber, combined to backscattered electron imaging of fixed (chemically or by quick freezing followed by freeze substitution and resin embedded samples is bringing an exponential amount of valuable information. In HIM there is no need of conductive coating and the depth of field is much higher than in any field emission SEM. As for FIB- and MSS-SEM, high resolution 3-D models of areas and volumes larger than any other technique allows can be obtained. The main results achieved with all these technological tools and some protocols for sample preparation are included in this review. In addition, we included some results obtained with environmental/low vacuum scanning microscopy and cryo-scanning electron microscopy, both promising, but not yet largely employed SEM modalities.

Effect of Piper betle on Giardia intestinalis infection in vivo.

Piper betle has been used as a medicinal plant in traditional medical systems throughout South and South East Asia. Experimental studies have revealed its wide and diverse biological and pharmacological effects. In this study, antigiardial activity of Piper betle was tested using experimental infections of Giardia intestinalis, the most common cause of protozoal diarrhoea worldwide, in Mongolian gerbils. Plants were extracted in water, methanol and methanol:tetrahydrofuran. Gerbils were treated for ten days intragastrically twice a day, with the dose of 40 mg of the extract per 100 g of body weight. Drug metronidazole was used as a negative control. Gerbils' faeces were taken every day and examined by flotation method, the number of shed cysts were counted using a haemocytometer. After gerbils' sacrifice and dissection, their duodena were then processed for examination using histological sectioning and scanning electron microscopy. The antigiardial activity was evaluated by the course of cyst shedding throughout the entire experiment. A significant decline in cyst shedding, evaluated by linear regression was found in gerbils treated with the aqueous extract. Our results indicate that the aqueous extract of P. betle shows giardicidal effects.

Coproscopy and molecular screening for detection of intestinal protozoa.

BACKGROUND: Intestinal parasitosis is one of several health concerns about immigrants who travel from endemic to non-endemic regions. Reliable rapid sensitive diagnostic tools, for use in non-endemic regions, are urgently required to enable frequent assessment of immigrant workers in jobs where risk of local transmission is a particular concern (e.g. food-handlers). We assessed the burden of intestinal protozoa in newly arrived immigrants and those applying for renewal of work permits in Qatar (n = 735), by both microscopic examination of stool samples and by Real Time PCR methodology. RESULTS: Prevalence was considerably higher using RT-PCR compared with coproscopy (Blastocystis hominis: 65.2 vs 7.6%; Giardia duodenalis: 14.3 vs 2.9%; Entamoeba histolytica: 1.6 vs 1.2%). Dientamoeba fragilis was sought only by RT-PCR (prevalence of 25.4%). Prevalence of G. duodenalis was significantly higher in male subjects, associated with blue collar workers and declined over time. Prevalence of B. hominis varied significantly with region of origin of subjects with highest values recorded among African immigrants. Prevalence of D. fragilis also varied with region of origin of subjects, and was lower in young female subjects and in renewal applicants compared with first-time applicants for work permits. CONCLUSIONS: We strongly recommend that, henceforth, intestinal protozoa should be screened by RT-PCR, with a particular focus on frequent assessment of immigrant food-handlers.

Giardia duodenalis induces pathogenic dysbiosis of human intestinal microbiota biofilms.

Giardia duodenalis is a prevalent cause of acute diarrheal disease worldwide. However, recent outbreaks in Italy and Norway have revealed a link between giardiasis and the subsequent development of chronic post-infectious irritable bowel syndrome. While the mechanisms underlying the causation of post-infectious irritable bowel syndrome remain obscure, recent findings suggest that alterations in gut microbiota communities are linked to the pathophysiology of irritable bowel syndrome. In the present study, we use a laboratory biofilm system to culture and enrich mucosal microbiota from human intestinal biopsies. Subsequently, we show that co-culture with Giardia induces disturbances in biofilm species composition and biofilm structure resulting in microbiota communities that are intrinsically dysbiotic - even after the clearance of Giardia. These microbiota abnormalities were mediated in part by secretory-excretory Giardia cysteine proteases. Using in vitro cell culture and germ-free murine infection models, we show that Giardia-induced disruptions of microbiota promote bacterial invasion, resulting in epithelial apoptosis, tight junctional disruption, and bacterial translocation across an intestinal epithelial barrier. Additionally, these dysbiotic microbiota communities resulted in increased activation of the Toll-like receptor 4 signalling pathway, and overproduction of the pro-inflammatory cytokine IL-1beta in humanized germ-free mice. Previous studies that have sought explanations and risk factors for the development of post-infectious irritable bowel syndrome have focused on features of enteropathogens and attributes of the infected host. We propose that polymicrobial interactions involving Giardia and gut microbiota may cause persistent dysbiosis, offering a new interpretation of the reasons why those afflicted with giardiasis are predisposed to gastrointestinal disorders post-infection.

Foodborne Giardiasis: Is There Any Relationship Between food Handlers and Transmission of Giardia duodenalis?

BACKGROUND: The foodborne diseases are amongst the main causes of morbidity and mortality in the human communities. Giardia duodenalis, the causative agent of giardiasis, is one of the foodborne parasites, which has public health importance. The aim of the current study was to assess the prevalence of G. duodenalis among food handlers in Andimeshk County, southwest of Iran. MATERIALS AND METHODS: This cross-sectional study was undertaken among 480 food handlers in 2015. The collected stool specimens were investigated using direct saline smear, Lugol's iodine- staining, and sucrose flotation methods. RESULTS: The overall prevalence of G. duodenalis in the examined participants was 12 (2.5%). The higher prevalence 75% (9/12) was found among participants with medium and low levels of education and 25% (3/12) belonged to those with high level of education. Direct microscopic examinations revealed two (0.4%) positive cases infected with Hymenolepis nana, with one of them showing mixed infection with G. duodenalis. CONCLUSION: Based on the obtained results, infected food handlers could be a potential source of intestinal parasitic infections, and transmission can occur through contaminated food. Therefore, we suggest that food handlers training programs should be implemented to increase the awareness of food handlers and reduce the transmission of intestinal parasites.