The sole known heme enzyme of the parasitic protist Giardia intestinalis is a flavohemoglobin (gFlHb) that acts as a nitric oxide dioxygenase (NOD) and protects the organism from the free radical nitric oxide. To learn more about the properties of this enzyme, we measured its nitric oxide dioxygenase, NADH oxidase, and cytochrome c reductase activities and compared these to the activities of the E. coli flavohemoglobin (Hmp). The turnover number for the NOD activity of gFlHb (23 s-1) is about two-thirds of that of Hmp (34 s-1) at pH 6.5 and 37 °C. The two enzymes differ in their sensitivity towards molecules that act as heme ligands. For both gFlHb and Hmp, inhibition with miconazole, a large imidazole ligand, is adequately described by simple competitive inhibition, with KI = 10 μM and 0.27 μM for gFlHb and Hmp, respectively. Inhibition plots with the small ligand imidazole were biphasic, which is consistent with previous experiments with carbon monoxide as a probe that show that the active site of flavohemoglobins exists in two conformations. Interestingly, the largest difference is observed with nitrite, which, like imidazole, also shows a biphasic inhibition plot; however, nitrite inhibits gFlHb at sub-millimolar concentrations while Hmp is not significantly affected. NADH oxidase activity measured under aerobic conditions in the absence of nitric oxide for Hmp was more than twice the activity of gFlHb. The addition of 1 mM hydrogen peroxide in these assays stimulated the NADH oxidase activity of gFlHb but not Hmp. Both enzymes had nearly identical cytochrome c reductase activities but the extent of the contribution of indirect reduction by flavohemoglobin-generated superoxide was much lower with gFlHb (4% SOD-inhibited) than with Hmp (17% SOD-inhibited). Although the active sites of the two enzymes share the same highly conserved residues that are important for catalysis, differences in the distal ligand binding site may account for these differences in activity and sensitivity towards NOD inhibitors. The differences observed in the NADH oxidase and cytochrome c reductase assays suggest that gFlHb may have evolved to protect the protist, which lacks both superoxide dismutase and catalase, from the damaging effects of superoxide by minimizing its production and from peroxide by actively reducing it.

Diarrheal diseases are the second leading cause of death in children worldwide. Epidemiological studies show that co-infection with Giardia intestinalis decreases the severity of diarrhea. Here, we show that Giardia is highly prevalent in the stools of asymptomatic school-aged children. It orchestrates a Th2 mucosal immune response, characterized by increased antigen-specific Th2 cells, IL-25, Type 2-associated cytokines, and goblet cell hyperplasia. Giardia infection expanded IL-10-producing Th2 and GATA3+ Treg cells that promoted chronic carriage, parasite transmission, and conferred protection against Toxoplasma gondii-induced lethal ileitis and DSS-driven colitis by downregulating proinflammatory cytokines, decreasing Th1/Th17 cell frequency, and preventing collateral tissue damage. Protection was dependent on STAT6 signaling, as Giardia-infected STAT6-/- mice no longer regulated intestinal bystander inflammation. Our findings demonstrate that Giardia infection reshapes mucosal immunity toward a Type 2 response, which confers a mutualistic protection against inflammatory disease processes and identifies a critical role for protists in regulating mucosal defenses.

Giardiasis is the most common intestinal parasitic disease worldwide. Clinical presentation ranges from asymptomatic to abdominal pain, diarrhea, and iron deficiency anemia. Treatment modalities include tinidazole, metronidazole, and paromomycin. We present a case of an adult man with anemia and suspected gastrointestinal bleeding who was found to have a duodenal nodule consistent with Brunner gland hyperplasia, and biopsy also showed Giardia. Limited case reports of Giardia diagnosed by duodenal biopsy are found in the literature. To the best of our knowledge, this is the first report of giardiasis presenting as Brunner gland hyperplasia.

Giardiasis is a small intestinal disease caused by the zoonotic parasite, Giardia duodenalis. This study presents the molecular findings of G. duodenalis infection in companion dogs, domestic livestock and wildlife in the Northern Jordan Basin, Israel. Identification of G. duodenalis was accomplished by nested PCR (nPCR) targeting the 18S rRNA gene. Samples were collected from water (five samples from four sources of which one was recycled water), as well as feces from wolves (Canis lupus) (n = 34), jackals (Canis aureus) (n = 24), wild boars (Sus scrofa) (n = 40), cattle (Bos taurus) (n = 40), dogs (Canis lupus familiaris) (n = 37) and nutria (Mayocastor coypus) (n = 100). All positive samples were sequenced and a phylogenetic tree was drawn using the Bayesian Inference (BI) algorithm. Differences in G. duodenalis prevalence between the different hosts were analyzed by Pearson\'s chi-square (p < 0.05). Of the total 275 fecal samples, 36 were positive for G. duodenalis (13%). Frequency rates among different animal species was highest in wolves (32.3%), whilst rates in wild boars (22.5%), dogs (16.2%), cattle (12.5%) and jackals (4.2%), were observed to be significantly lower (p < 0.001). Three out of 5 recycled water (RW) samples were G. duodenalis positive. Three clusters with high posterior probabilities (PP) were found in the BI: Cluster 1: samples from wolves, wild boars, water and cattle together with database sequences of assemblages A, B and F, Cluster 2: samples from dogs, nutria and a jackal with sequences from assemblage D and Cluster 3: samples from cattle, wild boars, wolves and dogs with sequences from assemblage C and D. We suggest that wolves serve as reservoirs of G. duodenalis in this region. The finding of Giardia in RW suggests that this vehicle may further contaminate crops intended for human consumption as this water source is used for agricultural irrigation.

Cryptosporidium spp., Giardia spp. and Enterocytozoon bieneusi are common zoonotic pathogens in humans and animals. Although rodents are important parts of the ecosystem and common hosts for these pathogens, little is known of the distribution, genetic diversity and zoonotic potential of these pathogens in wild rodents. A total of 442 fecal samples were collected from eleven wild rodent species in three provinces of China, and analyzed for these pathogens by PCR and DNA sequencing. The infection rates of Cryptosporidium spp., Giardia spp. and E. bieneusi were 19.9% (88/442), 19.8% (75/378) and 12.2% (54/442), respectively. Altogether, 23 known Cryptosporidium species/genotypes were identified and their distribution varied among different sampling locations or rodent species. Subtyping of the zoonotic Cryptosporidium species identified two novel subtype families XVe and XVf in C. viatorum, the subtype family XIIh and a novel subtype family XIIj in C. ubiquitum, and the subtype family IId in C. parvum. Three Giardia species were identified, including G. microti (n = 57), G. muris (n = 15) and G. duodenalis (n = 3), with G. duodenalis assemblages A and G identified in brown rats in urban areas of Guangdong. In addition, 13 E. bieneusi genotypes including eight known and five novel ones were identified, belonging to Groups 1, 2, 10, 14 and 15. Within nine genotypes in the zoonotic Group 1, common human-pathogenic genotypes D, Type IV, PigEbITS7 and Peru8 were detected only in brown rats and Lesser rice-field rats in urban areas of Guangdong. Apparent host adaptation and geographical differences were observed among Cryptosporidium spp., Giardia spp. and E. bieneusi genotypes in wild rodents in the present study. Furthermore, the zoonotic Cryptosporidium species and E. bieneusi genotypes commonly found here suggest a high zoonotic potential of these pathogens in wild rodents, especially in brown rats in urban areas. Hygiene and One Health measures should be implemented in urban streets and food stores to reduce the possible direct and indirect transmission of these rodent-related pathogens.

Giardia duodenalis (syn. G. intestinalis or G. lamblia) is a parasitic protozoan that infects the upper intestinal tract of a broad range of hosts, including humans and domestic animals. Thus, it has raised concerns about the public health risk due to companion animals. Recently, with the improvement of living standards and increasing contacts between pets and humans, the zoonotic transmission of Giardia has dramatically increased. From a genetic point of view, G. duodenalis should be viewed as a complex species that includes eight different species-specific genetic assemblages. The laboratory diagnosis is mainly based on the finding of microscopic cysts in stool samples by coprological examination. Other methods include the detection of antigens, immunoassays or PCR protocols, which allow the identification of Giardia assemblages. The study aimed to compare the performance of Direct Fluorescence Antibody test (DFA), zinc sulfate flotation technique (ZnSO4), rapid diagnostic test (RDT), end-point PCR amplification (PCR) for the detection of Giardia and to identify the concerning assemblages in a canine population from Central Italy. Direct fluorescence antibody test is the reference standard for laboratory diagnosis of Giardia in fecal samples from dogs, despite the microscopic examination after flotation remains the most useful method in many veterinary diagnostic centers. The present findings demonstrate the high performance of DFA and ZnSO4 in detecting Giardia, while RDT may be useful as alternative or complementary method to the DFA and ZnSO4. PCR performance was low, but it allowed determining Giardia B zoonotic assemblage in 25% of the PCR-positive specimens (15 out of 60), while the remaining PCR-positive isolates belonged to the dog-specific assemblage C. The 26% prevalence of G. duodenalis detected by DFA in owned dogs and the identification of potentially zoonotic assemblages underline the potential risk for public health and indicate frequent cross-species transmission of the parasite between humans and dogs.

Cryptosporidium and Giardia are protozoan parasites responsible for gastrointestinal illnesses in humans and in animal species. The main way these parasites are transmitted is by ingestion of their (oo)cysts in drinking water. Monitoring (oo)cysts in water sources is beneficial to evaluate the quality of raw water supplying treatment plants. Currently, the only standardized protocol to enumerate these parasites from water samples is United States Environmental Protection Agency (USEPA) Method 1623.1. With this method, we monitored three major water sources in Quebec over a year to assess temporal and geographical variations of these parasite (oo)cysts. These three water sources have independent watersheds despite being in the same region. We found a general pattern for Giardia, with high concentrations of cysts during cold and transition periods, and significantly lower concentrations during the warm period. Cryptosporidium\'s concentration was more variable throughout the year. Statistical correlations (Pearson\'s correlation coefficients) were established between the concentration of each parasite and various environmental parameters. The three study sites each showed unique factors correlating with the presence of both protozoa, supporting the idea that each water source must be seen as a unique entity with its own particular characteristics and therefore, must be monitored independently. Although some environmental parameters could be interesting proxies to the parasitic load, no parameter was strongly correlated throughout the whole sampling year and none of the parameters could be used as a single proxy for all three studies sources.

BACKGROUND: Enteric parasitic infections remain a major public health problem globally. Cryptosporidium spp., Cyclospora spp. and Giardia spp. are parasites that cause diarrhea in the general populations of both developed and developing countries. Information from molecular genetic studies on the speciation of these parasites and on the role of animals as vectors in disease transmission is lacking in Ghana. This study therefore investigated these diarrhea-causing parasites in humans, domestic rats and wildlife animals in Ghana using molecular tools.
METHODS: Fecal samples were collected from asymptomatic school children aged 9-12 years living around the Shai Hills Resource Reserve (tourist site), from wildlife (zebras, kobs, baboons, ostriches, bush rats and bush bucks) at the same site, from warthogs at the Mole National Park (tourist site) and from rats at the Madina Market (a popular vegetable market in Accra, Ghana. The 18S rRNA gene (18S rRNA) and 60-kDa glycoprotein gene (gp60) for Cryptosporidium spp., the glutamate dehydrogenase gene (gdh) for Giardia spp. and the 18S rDNA for Cyclospora spp. were analyzed in all samples by PCR and Sanger sequencing as markers of speciation and genetic diversity.
RESULTS: The parasite species identified in the fecal samples collected from humans and animals included the Cryptosporidium species C. hominis, C. muris, C. parvum, C. tyzzeri, C. meleagridis and C. andersoni; the Cyclopora species C. cayetanensis; and the Gardia species, G. lamblia and G. muris. For Cryptosporidium, the presence of the gp60 gene confirmed the finding of C. parvum (41%, 35/85 samples) and C. hominis (29%, 27/85 samples) in animal samples. Cyclospora cayetanensis was found in animal samples for the first time in Ghana. Only one human sample (5%, 1/20) but the majority of animal samples (58%, 51/88) had all three parasite species in the samples tested.
CONCLUSIONS: Based on these results of fecal sample testing for parasites, we conclude that animals and human share species of the three genera (Cryptosporidium, Cyclospora, Giardia), with the parasitic species mostly found in animals also found in human samples, and vice-versa. The presence of enteric parasites as mixed infections in asymptomatic humans and animal species indicates that they are reservoirs of infections. This is the first study to report the presence of C. cayetanensis and C. hominis in animals from Ghana. Our findings highlight the need for a detailed description of these parasites using high-throughput genetic tools to further understand these parasites and the neglected tropical diseases they cause in Ghana where such information is scanty.

Giardiosis remains one of the most prevalent enteric parasitic infections globally. Earlier molecular-based studies conducted in Egypt have primarily focused on paediatric clinical populations and most were based on single genotyping markers. As a result, there is limited information on the frequency and genetic diversity of G. duodenalis infections in individuals of all age groups.
Individual stool samples (n = 460) from outpatients seeking medical care were collected during January-December 2021 in Kafr El-Sheikh governorate, northern Egypt. Initial screening for the presence of G. duodenalis was conducted by coprological examination. Microscopy-positive samples were further confirmed by real-time PCR. A multilocus sequence typing approach targeted amplification of the glutamate dehydrogenase (gdh), beta-giardin (bg), and triose phosphate isomerase (tpi) genes was used for genotyping purposes. A standardised epidemiological questionnaire was used to gather basic sociodemographic and clinical features of the recruited patients.
Giardia duodenalis cysts were observed in 5.4% (25/460, 95% CI: 3.6-7.9) of the stool samples examined by conventional microscopy. The infection was more frequent in children under the age of 10 years and in individuals presenting with diarrhoea but without reaching statistical significance. Stool samples collected during the winter period were more likely to harbour G. duodenalis. All 25 microscopy-positive samples were confirmed by real-time PCR, but genotyping data was only available for 56.0% (14/25) of the isolates. Sequence analyses revealed the presence of assemblages A (78.6%, 11/14) and B (21.4%, 3/14). All assemblage A isolates were identified as sub-assemblage AII, whereas the three assemblage B sequences belonged to the sub-assemblage BIII. Patients with giardiosis presenting with diarrhoea were more frequently infected by the assemblage A of the parasite.
This is one of the largest epidemiological studies evaluating G. duodenalis infection in individuals of all age groups in Egypt. Our molecular data suggest that G. duodenalis infections in the surveyed population are primarily of anthropic origin. However, because assemblages A and B are zoonotic, some of the infections identified can have an animal origin. Additional investigations targeting animal (domestic and free-living) and environmental (water) samples are warranted to better understand the epidemiology of giardiosis in Egypt.

Giardia is a prevalent single-celled microaerophilic intestinal parasite causing diarrheal disease and significantly impacting global health. Double diploid (essentially tetraploid) Giardia trophozoites have presented a formidable challenge to the development of molecular genetic tools to interrogate gene function. High sequence divergence and the high percentage of hypothetical proteins lacking homology to proteins in other eukaryotes have limited our understanding of Giardia protein function, slowing drug target validation and development. For more than 25 years, Giardia A and B assemblages have been readily amenable to transfection with plasmids or linear DNA templates. Here, we highlight the utility and power of genetic approaches developed to assess protein function in Giardia, with particular emphasis on the more recent clustered regularly interspaced palindromic repeats/Cas9-based methods for knockdowns and knockouts. Robust and reliable molecular genetic approaches are fundamental toward the interrogation of Giardia protein function and evaluation of druggable targets. New genetic approaches tailored for the double diploid Giardia are imperative for understanding Giardia\'s unique biology and pathogenesis.