Giardia duodenalis is a common flagellated protozoan parasite that infects the small intestine of a wide range of vertebrate hosts. This study aimed to determine whether tracing of G. duodenalis isolates by current genetic typing tools is possible using an exemplary set of samples from infected cattle, buffalo and children from the Ismailia province, Egypt.

Background & Aims The protozoa Giardia duodenalis is a major cause of gastrointestinal illness worldwide, but underlying pathophysiological mechanisms remain obscure, partly due to the absence of adequate cellular models. We aimed at overcoming these limitations and recapitulating the authentic series of pathogenic events in the primary human duodenal tissue by using the human organoid system. Methods We established a compartmentalized cellular transwell system with electrophysiological and barrier properties akin to duodenal mucosa and dissected the events leading to G. duodenalis-induced barrier breakdown by functional analysis of transcriptional, electrophysiological, and tight junction components. Results Organoid-derived cell layers of different donors showed a time- and parasite load-dependent leak flux indicated by collapse of the epithelial barrier upon G. duodenalis infection. Gene set enrichment analysis suggested major expression changes, including gene sets contributing to ion transport and tight junction structure. Solute carrier family 12 member 2 and cystic fibrosis transmembrane conductance regulator-dependent chloride secretion was reduced early after infection, while changes in the tight junction composition, localization, and structural organization occurred later as revealed by immunofluorescence analysis and freeze fracture electron microscopy. Functionally, barrier loss was linked to the adenosine 3′,5′-cyclic monophosphate (cAMP)/protein kinase A-cAMP response element-binding protein signaling pathway. Conclusions Data suggest a previously unknown sequence of events culminating in intestinal barrier dysfunction upon G. duodenalis infection during which alterations of cellular ion transport were followed by breakdown of the tight junctional complex and loss of epithelial integrity, events involving a cAMP/protein kinase A-cAMP response element-binding protein mechanism. These findings and the newly established organoid-derived model to study G. duodenalis infection may help to explore new options for intervening with disease and infection, in particular relevant for chronic cases of giardiasis.

The small intestinal epithelium is the primary route of infection for many protozoan parasites. Understanding the mechanisms of infection, however, has been hindered due to the lack of appropriate models that recapitulate the complexity of the intestinal epithelium. Here, we describe an in vitro platform using stem cell-derived intestinal organoids established for four species that are important hosts of Apicomplexa and other protozoa in a zoonotic context: human, mouse, pig and chicken. The focus was set to create organoid-derived monolayers (ODMs) using the transwell system amenable for infection studies, and we provide straightforward guidelines for their generation and differentiation from organ-derived intestinal crypts. To this end, we reduced medium variations to an absolute minimum, allowing generation and differentiation of three-dimensional organoids for all four species and the subsequent generation of ODMs. Quantitative RT-PCR, immunolabeling with antibodies against marker proteins as well as transepithelial-electrical resistance (TEER) measurements were used to characterize ODM’s integrity and functional state. These experiments show an overall uniform generation of monolayers suitable for Toxoplasma gondii infection, although robustness in terms of generation of stable TEER levels and cell differentiation status varies from species to species. Murine duodenal ODMs were then infected with T. gondii and/or Giardia duodenalis, two parasites that temporarily co-inhabit the intestinal niche but have not been studied previously in cellular co-infection models. T. gondii alone did not alter TEER values, integrity and transcriptional abundance of tight junction components. In contrast, in G. duodenalis-infected ODMs all these parameters were altered and T. gondii had no apparent influence on the G. duodenalis-triggered phenotype. In conclusion, we provide robust protocols for the generation, differentiation and characterization of intestinal organoids and ODMs from four species. We show their applications for comparative studies on parasite-host interactions during the early phase of a T. gondii infection but also its use for co-infections with other relevant intestinal protozoans.

Giardiasis is an important gastrointestinal parasitic disease in humans and other mammals caused by the protozoan Giardia duodenalis. This species complex is represented by genetically distinct groups (assemblages A-H) with varying zoonotic potential and host preferences. Wild rodents can harbor potentially zoonotic assemblages A and B, and the rodent-specific assemblage G. Other Giardia spp. found in these animals are Giardia muris and Giardia microti. For the latter, only limited information on genetic typing is available. It has been speculated that wild rodents might represent an important reservoir for parasites causing human giardiasis. The aim of this study was to investigate the occurrence and distribution of Giardia spp. and assemblage types in wild rodents from different study sites in Germany. Screening of 577 wild rodents of the genera Apodemus, Microtus and Myodes, sampled at eleven study sites in Germany, revealed a high overall Giardia prevalence. Giardia species determination at the SSU rDNA gene locus revealed that Apodemus mice, depending on species, were predominantly infected with one of two distinct G. muris sequence types. Giardia microti was the predominant parasite species found in voles of the genera Microtus and Myodes. Only a few animals were positive for potentially zoonotic G. duodenalis. Subtyping at the beta-giardin (bg) and glutamine dehydrogenase (gdh) genes strongly supported the existence of different phylogenetic subgroups of G. microti that are preferentially harbored by distinct host species. The present study highlights the preference of G. muris for Apodemus, and G. microti for Microtus and Myodes hosts and argues for a very low prevalence of zoonotic G. duodenalis assemblages in wild rodents in Germany. It also provides evidence that G. muris and G. microti subdivide into several phylogenetically distinguishable subgroups, each of which appears to be preferentially harbored by species of a particular rodent host genus. Finally, the study expands the database of sequences relevant for sequence typing of G. muris and G. microti isolates which will greatly help future analyses of these parasites’ population structure.

Cryptosporidium spp. and Giardia spp. are important diarrhea-causing protozoan parasites worldwide that exhibit broad host ranges. Wild small mammals can harbor host-adapted and potentially zoonotic species of both parasites. The aim of this study was to investigate Cryptosporidium spp. and Giardia spp. in wild rodents and shrews in Portugal, focusing on the protist’s occurrence and genetic diversity. Molecular screening by PCR at the small subunit (SSU) rRNA gene locus of 290 fecal samples from wood mice (Apodemus sylvaticus), southwestern water voles (Arvicola sapidus), Cabrera’s voles (Microtus cabrerae), Lusitanian pine voles (Microtus lusitanicus), Algerian mice (Mus spretus) and greater white-toothed shrews (Crocidura russula) in Northeast Portugal revealed the low occurrence of Cryptosporidium spp. (1%) and high occurrence of Giardia spp. (32.8%). The analysis revealed that “species” was the only significant factor associated with the increasing probability of Giardia spp. infection, with the highest prevalence reported in southwestern water voles and Lusitanian pine voles. Cryptosporidium and Giardia species determination at the SSU rRNA gene locus revealed C. muris and G. microti as the only circulating species, respectively. Subtyping of the glutamate dehydrogenase (gdh) and beta-giardin (bg) genes provided evidence of the high genetic diversity within the G. microti clade. This study suggests that rodent-adapted G. microti occurs to a large extent in cricetid hosts and supports the limited role of wild rodents and shrews as natural sources of human infections in Northeast Portugal regarding the investigated parasites. Moreover, this is the first record of G. microti in southwestern water voles, Lusitanian pine voles, Algerian mice, wood mice and Cabrera’s voles and C. muris in Cabrera’s voles. Finally, this study improves the database of sequences relevant for the sequence typing of G. microti strains and provides new insights about the epidemiology of Giardia spp. and Cryptosporidium spp. in wild rodents and shrews, two parasite genera of high importance for public and animal health.