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About the Authors: Lenka Cernikova Affiliation: Laboratory of Molecular Parasitology, Institute of Parasitology, University of Zurich (ZH), Zurich, Switzerland Carmen Faso Affiliation: Laboratory of Molecular Parasitology, Institute of Parasitology, University of Zurich (ZH), Zurich, Switzerland ORCID logo http://orcid.org/0000-0002-1831-9365 Adrian B. Hehl * E-mail: adrian.hehl@uzh.ch Affiliation: Laboratory of Molecular Parasitology, Institute of Parasitology, University of Zurich (ZH), Zurich, Switzerland ORCID logo http://orcid.org/0000-0002-2110-4445 Citation: Cernikova L, Faso C, Hehl AB (2018) Five facts about Giardia lamblia. Based on the latest figures provided by WHO, G. lamblia is the third most common agent of diarrheal disease worldwide with over 300 million reported cases per annum, preceded only by rotavirus and Cryptosporidium parvum and hominis in the most vulnerable target group of children under five years of age [2]. ER, endoplasmic reticulum; GILRP, giardial putative low-density lipoprotein receptor; LDL, low-density lipoprotein; PM, plasma membrane; PV, peripheral vacuole; TEM, transmission electron microscopy; VSP, variant surface protein. https://doi.org/10.1371/journal.ppat.1007250.g001 Fact 4: G. lamblia survives in the environment as infectious cysts Completion of the life cycle by transmission of G. lamblia to a new host requires no vectors and is based on the alternation of a vegetative stage, the trophozoite, and an environmentally resistant infectious stage—the cyst. Novel rational design-based vaccination strategies against G. lamblia are yielding encouraging results Currently, treatment of giardiasis in humans is based almost exclusively on administration of antiprotozoals belonging to the family of 5-nitroimidazoles, whereas infected animals are treated with benzimidazoles.

Techniques for sub-classifying morphologically identical Giardia duodenalis trophozoites have included comparisons of the electrophoretic mobility of enzymes and of chromosomes, and sequencing of genes encoding β-giardin, triose phosphate isomerase, the small subunit of ribosomal RNA and glutamate dehydrogenase. To date, G. duodenalis organisms have been sub-classified into eight genetic assemblages (designated A–H). Genotyping of G. duodenalis organisms isolated from various hosts has shown that assemblages A and B infect the largest range of host species, and appear to be the main (or possibly only) G. duodenalis assemblages that undeniably infect human subjects. In at least some cases of assemblage A or B infection in wild mammals, there is suggestive evidence that the infection had resulted from environmental contamination by G. duodenalis cysts of human origin. Les techniques pour sous-classer morphologiquement des trophozoïtes identiques de Giardia duodenalis ont inclus des comparaisons de la mobilité électrophorétique des enzymes et des chromosomes et le séquençage des gènes codant pour la β-giardine, la triose-phosphate isomérase, la petite sous-unité ribosomique de l’ARN et la glutamate déshydrogénase. À ce jour, G. duodenalis a été sous-classé en 8 assemblages génétiques (désignés par A-H). Le génotypage de G. duodenalis isolés à partir de divers hôtes a montré que les assemblages A et B infectent le grand plus grand nombre d’espèces d’hôtes, et semblent être les assemblages principaux (ou peut-être uniques) qui infectent les sujets humains de manière indéniable. Dans au moins certains cas d’infection chez les mammifères sauvages par les assemblages A ou B, des éléments indiquent que l’infection était due à la contamination de l’environnement par des kystes de G. duodenalis d’origine humaine.

The genome of the eukaryotic protist Giardia lamblia, an important human intestinal parasite, is compact in structure and content, contains few introns or mitochondrial relics, and has simplified machinery for DNA replication, transcription, RNA processing, and most metabolic pathways. Protein kinases comprise the single largest protein class and reflect Giardia's requirement for a complex signal transduction network for coordinating differentiation. Lateral gene transfer from bacterial and archaeal donors has shaped Giardia's genome, and previously unknown gene families, for example, cysteine-rich structural proteins, have been discovered. Unexpectedly, the genome shows little evidence of heterozygosity, supporting recent speculations that this organism is sexual. This genome sequence will not only be valuable for investigating the evolution of eukaryotes, but will also be applied to the search for new therapeutics for this parasite.

Sexual recombination is a hallmark of eukaryotic evolution. Without recombination, asexual eukaryotes should succumb to deleterious mutations and more rapidly evolving pathogens. Giardia duodenalis , a parasitic protist, sits within one of the earliest-branching eukaryotic lineages and has no known sexual stage. Whether Giardia are ‘ancient asexuals’ has been long explored but is unresolved. Here, we find clear evidence of sex in Giardia and also discover an asexual sublineage that has a broader host range than its sexual ancestor. This asexual lineage is not ancient, and is accumulating deleterious mutations. Unlike its sexual counterparts, its genetic variation lacks the signatures of selection and Red Queen coevolution. We propose a new hypothesis that explains how a mutational meltdown during Muller’s Ratchet might enable asexual pathogens to expand their host ranges transiently. Fittingly, our results suggest that Giardia is not the last exception to, but rather further evidence of, the essentiality of eukaryotic sex. Sexual reproduction is thought to be essential for long-term survival of eukaryotes. This study shows that Giardia, once suspected to be anciently asexual, retains evidence of sex while a newly derived asexual lineage is accumulating mutations and expanding its host range.

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.

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

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. Giardia duodenali s, Cryptosporidium spp. et Toxoplasma gondii sont des parasites protozoaires qui ont été soulignés comme agents pathogènes émergents dans les aliments par l’Organisation des Nations Unies pour l’alimentation et l’agriculture et l’Organisation Mondiale de la Santé. Selon l’Autorité Européenne de Sécurité des Aliments, 4786 épidémies d’origine alimentaire et hydrique ont été enregistrées en Europe en 2016, dont 0.4% ont été attribuées à des parasites, incluant Cryptosporidium , Giardia et Trichinella . Jusqu’en 2016, aucune méthode standardisée n’était disponible pour détecter les kystes de Giardia et les oocystes de Cryptosporidium et Toxoplasma dans les aliments. Aucune réglementation n’est donc proposée concernant ces dangers. Cependant, compte tenu de leur faible dose infectieuse, l’ingestion d’une quantité d’aliments faiblement contaminés peut entraîner une infection de l’homme. Pour évaluer le risque lié aux protozoaires dans les aliments, des efforts doivent être faits dans l’évaluation de l’exposition pour estimer la contamination le long de la chaîne alimentaire, depuis la matière première jusqu’aux consommateurs. Cette évaluation nécessite de déterminer   : (i) la prévalence de parasites infectieux dans les aliments, (ii) l’efficacité des mesures de maîtrise pour éliminer cette contamination. Pour mener une telle évaluation, des méthodes capables d’identifier des parasites viables (vivants) et infectieux sont requises. Cette revue décrit les méthodes actuellement disponibles permettant d’évaluer l’infectiosité et la viabilité des kystes de G. duodenalis et des oocystes de Cryptosporidium spp. et T. gondii, et leur potentiel pour être appliquées dans l’évaluation de l’exposition pour déterminer la présence de parasites infectieux et/ou caractériser l’efficacité des mesures de maîtrise. Les avantages et limites de chaque méthode sont présentés et une stratégie d’analyses est proposée pour évaluer l’exposition à ces protozoaires.

Background The two intestinal protozoan parasites Giardia duodenalis and Cryptosporidium parvum cause infections in a wide spectrum of vertebrates and have also been shown to infect suitable hosts simultaneously. To investigate potential effects between these parasites and on host cells, a co-infection model with IPEC-J2 cells was established. Methods Optimal infection conditions and several infection doses of both parasites were tested. The effect of Giardia growth medium on IPEC-J2 cells was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, while the effect of different infection doses of each parasite on host cell viability was investigated by CellTiter Blue cell viability assay. For co-infection, IPEC-J2 cells were first infected with C. parvum sporozoites, and 3.5 h later, G. duodenalis trophozoites were added. Parasite propagation during single infection and co-infection were analyzed by quantitative real-time polymerase chain reaction (qPCR) as well as immunofluorescent staining. Results The infection with C. parvum sporozoites had no significant impact on cell viability, while G. duodenalis trophozoites affected cell culture in a dose dependent manner. The amount of gene copies of C. parvum in single and co-infected cells did not differ significantly, while statistically higher amounts of G. duodenalis gene copies in co-infected cell cultures were identified. Conclusions In this study, single infections and co-infections of IPEC-J2 cells with C. parvum and G. duodenalis were established and optimized over a period of 72 h. Graphical Abstract

Differentiation into infectious cysts through the process of encystation is crucial for transmission and survival of the intestinal protozoan parasite Giardia intestinalis. Hitherto the majority of studies have focused on the early events, leaving late encystation poorly defined. In order to further study encystation, focusing on the later events, we developed a new encystation protocol that generates a higher yield of mature cysts compared to standard methods. Transcriptome changes during the entire differentiation from trophozoites to cysts were thereafter studied using RNA sequencing (RNA-seq). A high level of periodicity was observed for up- and down-regulated genes, both at the level of the entire transcriptome and putative regulators. This suggests the trajectory of differentiation to be coordinated through developmentally linked gene regulatory activities. Our study identifies a core of 13 genes that are consistently up-regulated during initial encystation. Of these, two constitute previously uncharacterized proteins that we were able to localize to a new type of encystation-specific vesicles. Interestingly, the largest transcriptional changes were seen in the late phase of encystation with the majority of the highly up-regulated genes encoding hypothetical proteins. Several of these were epitope-tagged and localized to further characterize these previously unknown genetic components of encystation and possibly excystation. Finally, we also detected a switch of variant specific surface proteins (VSPs) in the late phase of encystation. This occurred at the same time as nuclear division and DNA replication, suggesting a potential link between the processes.

Background Cryptosporidium parvum and Giardia duodenalis (assemblages A and B) are ubiquitously occurring protozoan parasites infecting a broad range of hosts. Co-infection with both parasites in suitable hosts have been reported, but information on structural or functional alterations in host cells caused by simultaneous infection is rare. Previous findings showing an enhanced replication of G. duodenalis during co-infection suggest synergistic effects of both parasites that were investigated in this in vitro study. Methods The tight junction proteins claudin ( CLDN) 1, 4, 6, and 7 as well as the glucose transporters ( GLUT) 1 and 2 of IPEC-J-2 cells were examined comparing single and co-infections on gene expression level after 24 h, 48 h, and 72 h post infection (p.i.). Additionally, an analysis of intracellular glucose levels was performed 48 h p.i. Results No significant changes of the gene expression of the examined tight junction proteins were observed. Regarding the glucose transporters, GLUT2 was significantly decreased in cells infected by C. parvum sporozoites compared to cells infected by G. duodenalis trophozoites 48 h p.i. ( p  = 0.017) as well as compared to uninfected control cells ( p  = 0.021). Additionally, co-infected cells showed a significantly increased intracellular glucose level ( p  = 0.022) and C. parvum infected cells a non-significant trend of an increased intracellular glucose level ( p  = 0.056) in comparison to control cells. Compared to G . duodenalis mono-infected cells, co-infected cells showed a tendency for higher intracellular glucose levels ( p  = 0.057). Conclusion Cryptosporidium parvum had an impact on GLUT2 transcript abundance and also increased glucose levels in mono- and co-infection under the tested conditions, while G. duodenalis did not alter the examined glucose transporter and tight junctions markers in this model. Graphical Abstract