Immunomagnetic separation (IMS) is a purification assay commonly utilised in isolating and enriching target microorganisms from complex samples. One such application of IMS is in the detection of Cryptosporidium and Giardia in water. Across the globe, water laboratories conduct routine testing following the guidelines provided by regulatory bodies such as the United States Environmental Protection Agency (USEPA) and the United Kingdom Drinking Water Inspectorate (DWI), where IMS is used in conjunction with immunofluorescence or IF staining and fluorescence microscopy analysis. One of the major problems encountered in IMS, and by extension IF analyses, is crossreactivity. Nonspecific binding to other particulate matter can lead to false positives in pathogen identification and inaccuracy in result reporting, which can have major socio-economic consequences in the larger scale. In this study, the relationship between IMS and IF analysis is assessed. Through flow cytometry analyses it has been identified that the antibodies conjugated to the magnetic particles in IMS kits and those fluorescently labelled in IF staining kits vary across commercially available kits. With processing water matrices of various complexities, such as highly turbid and chalky samples, it was demonstrated that the resulting IMS recoveries and the quality of the microscopy slides for visualisation are impacted by the antibodies used in the kits. Utilising IMS kits and IF staining kits that have highly specific antibodies to Cryptosporidium oocysts and Giardia cysts, respectively, would assist in reducing the problems with crossreactivity and nonspecific binding. Further investigations should be conducted in assessing the factors that affect IMS and IF, with the end goal of incorporating standardised approaches in processing complex water matrices for routine waterborne pathogen detection. 

The parasitic protist Giardia duodenalis causes in human and animal giardiasis, a diarrheal disease. Two G.duodenalis genetic groups (Assemblages A and B) cause human infection. High variability in the range of giardiasis symptoms is well documented and eventually linked to differences between and within Assemblages. Extracellular vesicles (EVs) operate as cargoes from cell to cell, for proteins and nucleic acids being implicated in physio-pathological processes. Here we characterize and compare EV proteomes from assemblage A and B. Released EVs, as well freely secreted proteins, were purified from spent medium by serial ultracentrifugation and morphologically and biochemically characterized by EM and DLS. EVs and secretome protein content were identified by HR-MS and datasets of relative abundance analyzed for protein-protein interaction networks and functional enrichment. Over 20% of Giardia's annotated proteome were identified extracellularly and ~4-fold more proteins than previous reports were quantifying. We unveiled a conserved extracellular proteome but with distinct signatures in assemblages A and B, consistent with variant pathophysiology and virulence. We mapped distinct protein signatures of Giardia EV sub-populations (i.e. exosomes and micro-vesicles) including (e.g. ribosome-mediated translation, cilium, vesicular trafficking, and exosome secretion). Mechanistic markers of ESCRT-dependent, ESCRT-independent, and Ectosomes pathways within the EV proteome were also identified. By exploring “conserved” and “eukaryotic-innovative” compositions in Giardia EV proteome relative to archaea EV proteome, we observed complex cellular processes providing insights into the origins of eukaryotic intercellular communication. Collectively, our findings advanced current comprehension of Giardia's protein release dynamics, establishing novel correlations between different Giardia Assemblages and diverse extracellular vesicle subtypes. 

Giardia intestinalis is divided into eight assemblages (A-H) which infect a wide range of mammals, including humans (A and B). The most widely studied G. intestinalis assemblage is AI (strain WB6). A reference genome, extensive transcriptomic and proteomic data sets exist for this assemblage. However, currently AI WB6 is not the most clinically relevant isolate from assemblage since the most common assemblage A variant in humans is sub-assemblage AII. AII isolates (like strains DH, AS98 and AS175) are distinguished from AI by host specificity, genetic repertoire and a much higher allelic sequence heterozygosity (ASH). Naturally, the question raises what causes higher ASH in AII and what mechanisms are responsible for the observed differences in genetic makeup between AI and AII. To tackle those questions, we plan to unveil the functions of genes that are involved in DNA repair and/or recombination throughout the parasite’s lifecycle stages, especially during encystation. The genes in question are known as meiosis-related genes and code for DNA topoisomerase (SPO11), synaptonemal complex protein (HOP1), recombinase (RAD51) and recombination-associated protein (MND1). After knocking these genes out in trophozoites using a Cas9/gRNA-mediated technique we shall investigate DNA repair efficiency, diplomixis (asexual process) and recombination (sexual process) in AI and AII assemblages. We predict differences in the temporal pattern of meiosis-related gene expression and in the recombination ability between distinct strains, as it is already established for other genes. 

Giardiasis, caused by the protozoan Giardia duodenalis, is a widespread parasitic infection affecting approximately 200 million people annually, predominantly in low- and middle-income countries. This infection is linked with undernutrition, enhancing the effects on intestinal dysfunction. Beyond its direct effects, Giardia infection exacerbates the consequences of malnutrition by impairing nutrient absorption and altering the gut microbial composition, abundance, and metabolism. These disruptions can result in enteropathies, impaired metabolic health, and stunted physical and cognitive development, particularly in children. Understanding the complex interplay between parasitic infections and host metabolic health is therefore critical to addressing the broader health implications of giardiasis. To investigate this, a mass spectrometry-based targeted metabolomics approach was used to analyse serum samples from Giardia-infected mice subjected to a protein-deficient diet. Our findings highlight that Giardia infection significantly impacts host-microbial metabolic interactions. One notable change was the decreased concentrations of p-cresyl sulfate in the serum of infected mice, indicating disruptions to the microbial metabolism of tyrosine and 4-hydroxyphenylacetate (4HPA). Reductions in circulating conjugated primary bile acids were also noted with infection, this included taurocholic acid and glycocholic acid. Bile acids have known antimicrobial and biochemical signaling properties. These changes may reflect increased gut microbial bile salt hydrolase (BSH) activity and enhanced dehydroxylation processes following infection. Collectively, these results provide novel insights into the metabolic derangements induced by Giardia infection, inducing perturbations at the microbiome level with downstream consequences for host metabolic homeostasis. 

During the past few decades, next-generation sequencing, particularly whole genome sequencing (WGS), has become a widely used method to study microorganisms of public health importance. Cryptosporidiosis, a disease caused by the protozoan parasite, Cryptosporidium, is the second leading cause of diarrhea in infants and one of the major contributors of diarrheal deaths among children under five in developing countries. Despite having such a huge impact, WGS of Cryptosporidium spp. has been difficult to implement due to experimental challenges. Obtaining pure Cryptosporidium DNA from clinical and environmental samples is difficult because the oocysts are shed into feces, making the purification process both difficult and expensive while yielding limited amounts of DNA (<40 fg/oocyst). The absence of optimized in vitro culture systems further complicates the purification process. Moreover, the extracted DNA is often heavily contaminated with host and microbial DNA, adding another layer of difficulty to achieving a pure sample suitable for downstream analyses. Thus, WGS of Cryptosporidium is constrained by limitations of implementing oocyst purification from large numbers of samples and the low yields of DNA from purified samples. To overcome the challenges of limited DNA availability, we present a library preparation protocol (iNextEra) for WGS that works across a broad range of input DNA amounts (<1 ng to >60 ng), requires little bench time and no specialized equipment, succeeds with limited PCR cycles, and yet is cost-efficient. To overcome the challenges associated with obtaining purified DNA, we also introduce a hybrid capture protocol (myBaits with CryptoCap_100K bait set) that can enrich Cryptosporidium DNA from a complex background, increasing the percentage of reads mapping to target Cryptosporidium reference genomes, thus reducing the amount of sequencing needed (thereby reducing sequencing costs). Both protocols have been validated and are actively used to process large-scale Cryptosporidium samples collected through collaborations across Asia, Africa, North America, Europe, and Australia. These efforts are aimed at investigating the global diversity of Cryptosporidium and advancing our understanding of cryptosporidiosis.

Cattle can shed large quantities of infectious Cryptosporidium and Giardia oo/cysts into the environment and are major revservoirs of zoonotic species. Understanding the species range and extent of genetic diversity of these parasites in cattle is essential to better understand and control zoonotic transmission. Globally, zoonotic cryptosporidiosis is mainly caused by C. parvum IIa subtypes, which are also the dominant subtypes in calves. In China however, IIa subtypes have not been reported in cattle, with the C. parvum IId subtype family responsible for all infections in cattle, which are also reported in humans in China. Molecular-based studies to date support calves as a major source of human C. parvum infections, however a widely used multi-locus sequence typing (MLST) tool and whole genome sequencing (WGS) is essential to confirm this. Our understanding of zoonotic transmission of Giardia from calves to humans is poorly understood due to issues with currently used typing systems and a recent taxonomic revision. New species-specific MLST tools have recently been developed and validated based on comparative Giardia genomics, but have yet to be extensively used. Well-designed epidemiological studies using improved typing tools are critical to to our understanding of the transmission dynamics of these important parasites. 

Nitroimidazole antimicrobials (e.g., metronidazole, tinidazole) are the first-line treatment for patients with giardiasis. Nitroimidazole-refractory giardiasis cases have been on the rise in the last two decades, particularly among travellers returning from south-east Asian countries. The interaction between Giardia duodenalis and the gut microbiota of the host plays an important role in the pathophysiology of giardiasis, but little is known about how the parasite impacts the human gut microbiome. In fact, few longitudinal studies exist, and none focuses on patients refractory to medical treatment. Here we present clinical and microbial community profiling by amplicon sequencing follow-up data on two patients with refractory giardiasis. A 31-year male and a 32-year female travelling to India experienced severe gastrointestinal symptoms during and immediately after the journey. Both were diagnosed with a G. duodenalis (assemblage B) infection. Treatment regimens with tinidazole 50 mg/kg/day, metronidazole 25 mg/kg/day, and quinacrine 50 mg/kg/day failed during a 129-day treatment period. Eight consecutive stool DNA samples (3 from the male, 5 from the female) spanning all treatment period were collected. Two post-infection negative samples (one from each patient) were collected more than one year later, when the symptoms have resolved. Gut microbiome composition was investigated using Illumina technology-based 16S rRNA gene profiling. A significant reduction in alpha diversity during infection was evidenced, which was sustained once infection was resolved. Post-infection gut microbial communities were different from those observed during infection, with an important individual effect. Changes in the relative abundances of bacterial taxa were observed at several ranks from phylum to genus. During infection, both subjects featured an increase of Prevotella, whereas Oscillibacter was the only common taxon identified at the post-infection stage in both patients. The male patient experienced a remarkable decrease of Ruminococcus taxa throughout the disease course and an increase of Flavobacterium ones after it. This is the first longitudinal bacterial community study in Giardia-infected adult refractory patients. We evidenced microbial shifts that may be associated with infection, treatment effect, or microbial restoration after infection, and can potentially be used to monitor disease evolution. Further studies involving larger cohorts are required to elucidate if they are a cause or a consequence of refractory giardiasis. 

Cryptosporidium can infect all mammals and is the pathogen causing diarrhoeal disease. Currently, no effective vaccine against cryptosporidiosis exists, and treatment strategy remains limited. Nitazoxanide, the only treatment approved by the Food and Drug Administration (FDA), is only effective in treating patients with normal immunocompetence and can shorten the duration of diarrhoea. However, it does not show therapeutic efficacy in infants, immunocompromised people and young animals at high risk of severe disease, so developing effective anti-cryptosporidium drugs is needed. In this study, we constructed an in vitro system for the evaluation of drug efficacy using Cryptosporidium parvum with a luciferase gene expression cassette and evaluated the antiprotozoal efficacy using existing drugs and compounds with reported drug efficacy in other Apicomplexa species. We discuss the utility of drug discovery research using luciferase-expressing parasites and future developments towards fully effective treatments for cryptosporidiosis.

Giardia infection poses significant public and veterinary health concerns worldwide, primarily transmitted through the fecal-oral route or indirectly by contaminated water or food. Very limited data exists on the diversity of Giardia assemblages infecting humans and animals in Norway. This ongoing study aims to determine assemblages and sub-assemblages of domestic and imported Giardia in Norway. Seven routine microbiological laboratories in Norway participated in the study. These have reported 243 human and 52 animal Giardia positive cases. Of these, 197 human samples, and 40 animal samples, were available for genotyping so far. Assemblage determination was done by nested PCR targeting the triosephosphate isomerase (tpi) gene, with subsequent sequencing. Domestic assemblage A isolates have been further genotyped using the 6-marker gene high-resolution multilocus sequence typing (MLST) strategy (Ankarlev et al. 2018). Data on the origins of human infections was obtained from Norwegian Surveillance System for Communicable Diseases. To date, 100 (50.8%) of the 197 Giardia isolates from human samples were successfully sequenced and showed predominance of assemblage B (59%), followed by assemblage A (40%), and assemblage E (1%). Sub-assemblage AII dominated, while sub-assemblages BIII and BIV were equally distributed. Assemblage distribution showed no differences between male (55.1%) and female (44.4%) patients. However, assemblage A was more common in people over 75 years old.  Out of 100 sequenced cases, 40% were imported, 27% were domestic, while data were unavailable for 33%. Imported cases were predominantly assemblage B (72%), while the domestic cases were primarily assemblage A (56%). Among 40 animal cases, 13 isolates (32.5%) were successfully sequenced. Assemblage B was found in a monkey, while eight were assemblage E (cattle/sheep), and 4 were assemblage A (3 dogs, 1 sheep).  This study provides the first comprehensive insights into Giardia assemblage distribution in Norway. Assemblage A strains seem commonly found in domestic cases, and these are being MLST genotyped to assess circulating strains and follow transmission pathways in Norway.  

Corsica, a mountainous Mediterranean island, relies significantly on livestock farming for its economic and cultural sustenance. Cryptosporidiosis, a widely distributed parasitic protozoan disease with a broad host range, including humans, bovines, canines, and horses, poses a significant concern. Despite the favorable context for such infections, the circulation of Cryptosporidium species remains unknown in Corsica, both in humans and animals. In this study, fecal samples were collected from various livestock animals on the island and analyzed using coproscopy and molecular biology techniques to identify Cryptosporidium species and subtypes. A total of 127 fecal samples from 109 livestock farms were examined, with 92% originating from small ruminant farms. The findings revealed that 96% of the animals were infected with at least one internal parasite, and Cryptosporidium spp. were identified in 8 fecal samples (6.3%) from 7 different farms (6.4% of the farms). Two zoonotic species, Cryptosporidium parvum and Cryptosporidium ubiquitum, were detected. The parasite was more prevalent in South Corsica than in Upper Corsica, with goats being the most affected species, accounting for 88% (7/8) of the positive animals. Additionally, two subtypes of C. parvum, IIdA14G1 and IIdA17G1, were identified. These findings highlight the circulation of Cryptosporidium in island livestock farms and its potential health impact on young animals. The identification of these zoonotic species underscores the exposure risk to human populations, despite the absence of reported human cases to date.

Context: Cryptosporidiosis is a gastrointestinal infection caused by Cryptosporidium spp., with transmission occurring through contaminated water, food, or direct contact with infected hosts. The oocysts of Cryptosporidium are highly resilient to environmental stresses, and the two predominant species, C. parvum and C. hominis, differ in transmission routes and ecological interactions. Understanding the surface properties of oocysts, such as the magnitude of the electrostatic repulsion/attraction forces, is crucial for investigating their stability and the dynamics of their dissemination in the environment.

Objective: This study aims to investigate the zeta potential values of oocysts from various Cryptosporidium species to explore how oocysts surface charge varies with pH, which could influence their adhesion to surfaces and their environmental persistence.

Methods: Cryptosporidium oocysts from 17 positive stool samples were purified using immunomagnetic separation. The subtypes included IbA10G2 (n=1), IbA13G3 (n=2), IdA16 (n=1), IeA11G3T3 (n=1), IfA12G1 (n=1), IIaA15G2R1 (n=7), IIaA17G1R1 (n=1), IIaA18R1 (n=2), and IIdA22G1 (n=1). Zeta potential was measured in suspensions of purified oocysts at three pH levels (acidic ≈2.5, neutral ≈6, and basic ≈12) using a Zetasizer Ultra (Malvern Panalytical, UK). Statistical comparisons of zeta potential values were conducted using ANOVA, with a significance threshold of p-value < 0.05.

Results: Zeta potential values varied significantly with pH. At neutral and basic pH, all subtypes exhibited negative zeta potential, while at acidic pH, a strong heterogeneity of zeta potential was observed. These results indicate that the isoelectric point differs among Cryptosporidium subtypes, and that pH is a critical factor in determining surface charge, with acidic conditions inducing substantial variability in zeta potential between subtypes.

Discussion: The observed pH-dependent changes in zeta potential align with previous findings showing that surface charge becomes more negative as pH increases [1]. The variation in zeta potential between subtypes suggests that Cryptosporidium oocysts may exhibit different behaviours in diverse environmental settings. These differences in surface charge may influence oocyst stability, aggregation, and interactions with surfaces, potentially affecting the persistence and dissemination of some Cryptosporidium subtypes in contaminated environments.

Giardia duodenalis is a cosmopolitan protozoan that infects mammals and is responsible for gastroenteritis. This species is subdivided into eight assemblages (A to H), which can only be distinguished through genetic approaches. Assemblages A (further divided into two sublineages A1 and A2) and B represent the most prevalent infectious lineages in humans. The typing of different strains remains underdeveloped, relying on a set of three loci (BG, TPI, and GDH). However, this strategy shows limitations for the molecular characterization of assemblage A strains due to low genetic diversity at these loci. Differentiating infectious lineages is essential for characterizing epidemic strains or distinguishing chronic infections from successive infections or mixed infections. In this study, we evaluated the combination of these three markers with the genotyping of six new markers (termed MLST). For this purpose, 48 human-derived Giardia strains (14 strains from the National Reference Center for Giardiasis in Rouen, France, 23 Ecuadorian strains, and 11 Ivorian strains) were utilized. Total DNA was extracted from raw feces. The loci were amplified using nested PCR and then sequenced using the Sanger method. The generated sequences were grouped by sample and compared with loci from reference strains. Fifty percent of the strains were successfully amplified across the nine loci, and 75% were amplified at least across eight loci. Phylogenetic trees reveal a much more precise resolution for the molecular analysis of assemblage A strains using the combination of the two sets of molecular markers, compared to the method based on the typing of three loci. In conclusion, this study demonstrates that the combined use of two sets of molecular markers allows for the accurate identification of infectious sublineages of Giardia.

Cryptosporidium and Giardia are protozoan parasites with diverse host ranges. Water acts as a vector for infective stages of both parasites, with human cases linked to swimming pools, natural recreational waterways and drinking water. Wastewater streams associated with humans may also act as a transmission source to non-human hosts. While many studies have examined the epidemiology of these parasites in terrestrial hosts, few have examined Cryptosporidium and Giardia in Australia’s marine ecosystems.   We have been investigating the prevalence and diversity of both Cryptosporidium and Giardia in little penguins (Eudyptula minor) and Australian sea lions (Neophoca cinerea). Faecal samples were acquired from individual penguins (n = 128) and sea lions (n = 147) in addition to host data such as age class, sex and weight. Prevalence ranged from 1% to 8.59% for both parasites in both penguins and seals. Isolated strains represented novel genotypes/species and those typically associated with human infection.   Moving beyond opportunistic sampling of wildlife allows for the relationship between parasite prevalence, diversity and host health to be defined. Overall, this project will inform management of threatened marine systems and provide data enabling evaluation of marine vertebrates as sentinels of ecosystem health in the Australian context  

The zoonotic potential of Giardia duodenalis of different assemblages has been debated up and down, and even recent publications take it as a given. However, unlike with Cryptosporidium, where cases of zoonotic transmission are well documented, there are very few cases where natural animal-to-human transmission seems highly probable. The only one with a very clear likelihood, is a case of giardiasis occurring in a toddler who ingested droppings from a pet chinchilla, which was subsequently found to be infected with Giardia.  Here we describe the case of a fieldworker who was assisting in a parasitology project by collecting faecal samples from wild reindeer in the mountains of Trøndelag County, Norway and carried on working - despite insufficient gloves - before stopping for a lunch break…..  The reindeer samples (N=41) collected were analysed for various parasites, including Giardia, and 18 were positive for Giardia cysts, of which many were categorised as being “high shedders” (>50 cysts per field of view). These results have been published. Some days after sample collection, the fieldworker developed diarrhoea; faecal samples delivered to the relevant GP were analysed in the local medical microbiology laboratory and giardiasis reported. The DNA eluate from the fieldworker’s sample was obtained from the laboratory and, along with Giardia samples from the reindeer, molecular characterisation was attempted.  At the GDH gene, both the reindeer sample and the fieldworker’s sample were found to be AI, with identical sequences. Due to the small amount of DNA available, characterisation of the Giardia from the human sample could not be continued – but the reindeer samples were further characterised, and at the RHP26 gene two samples were found to have identical sequences to that previously reported from a Giardia sample from a person (who had been considered likely to have been infected via a roe deer), and another sample had a sequence identical to that of a Giardia isolate from a moose.  Although both “wildlife” (of unknown species) and people have long been known to both be suitable hosts for Giardia Assemblage AI, convincing examples of transmission between these host types is generally thin. This case suggests, however, that those in contact with cervids and their faeces may be particularly exposed to this infection. 

Despite the significant public and veterinary health importance of Cryptosporidium and Giardia, data from certain regions, such as Armenia in the Caucasus, remain scarce. Limited studies have reported Cryptosporidium infections in livestock and humans in Armenia, but environmental contamination data are lacking. To address this gap, 24 raw water samples and two sediment samples were collected from rivers and lakes across nine regions of Armenia, including the capital cityt Yerevan. Sampling sites included agricultural and recreational areas, with water samples collected at 20–30 cm depth, 2–3 m from the banks, into clean 10 L containers. Sediment samples (approx. 100 g) were collected into clean plastic bags. The samples were analyzed using various methods, including modified Ziehl-Neelsen (mZN), Lugol stain, immunofluorescent antibody test (IFAT), qPCR, and immunomagnetic separation (IMS) with IFAT for sediment samples. Among 23 water samples analyzed by mZN, Cryptosporidium oocysts were detected in nine (39%), with concentrations ranging from 4 to 20 oocysts per 10 L. One sample was also positive for Giardia cysts by lugol stain (2 cysts per 10 L). Of two samples analyzed by IFAT, one was negative, and the other showed 18 Giardia cysts per 10 L. Of 11 water samples analyzed by qPCR, Cryptosporidium DNA was detected in only one sample, which had also tested positive by mZN. Both sediment samples analyzed by IMS and IFAT were positive for Cryptosporidium and Giardia. These findings suggest substantial contamination of raw water sources in Armenia and highlight the importance of further studies to better understand environmental contamination and its link to host infections in these catchment areas. 

Diarrheal disease is still an important cause of mortality in developing countries, where it mainly affects children under five years of age. In Cuba, although mortality from this cause has been reduced, it is necessary to know the contribution of intestinal parasites in the development of diarrhea, as well as to determine some clinical-epidemiological characteristics in hospitalized children. A study on intestinal parasitism was carried out in 113 children admitted to the "William Soler" Pediatric Teaching Hospital from April to November 2023. For this purpose, a stool sample was taken from each child, which was processed by three conventional parasitological methods (direct examination, Willis technique, and modified Zielh-Neelsen technique). DNA extraction was performed on all samples and real-time PCR was used to identify Giardia duodenalis and Cryptosporidium spp. The prevalence of intestinal parasite infection was 38.7%. The most representative species were G. duodenalis (15.8 0%), Blastocystis spp. (13.7%) and Cryptosporidium spp. (13.0 %).  No significant differences were found in terms of intestinal parasitic infection and the clinical and epidemiological characteristics evaluated, except persistent diarrhea (more than 14 days) caused by Cryptosporidium spp. Consumption of unboiled water and living in a rural area were risk factors for the development of intestinal parasitic infections. The use of real-time PCR for the diagnosis of cryptosporidiosis and giardiasis considerably increased diagnostic sensitivity compared to conventional techniques. These results illustrate the role that intestinal parasitosis, particularly cryptosporidiosis and giardiasis, can play within the broad etiological spectrum of diarrhea, and also show some epidemiological characteristics of parasitic infections in a group of Cuban children admitted with diarrhea.

Introduction: In Cuba, two National Surveys of Intestinal Parasites were carried out in the general population in 1984 and 2009, respectively, and even the general prevalence decreased from 65.1 % to 35.1 % from one study to the other, school-age children were the most affected group in both national surveys. [1] Therefore, we aimed to determine the current prevalence of intestinal parasites and risk factors in Cuban children aged 1-14 from all country provinces. Methods: A descriptive cross-sectional study was conducted from April 2023 to February 2024 on 19.000 children in the three regions of the country (eastern, central, and western). A stool sample was taken for each child, which was processed by three parasitological techniques (direct wet mount, flotation technique, and Kato-Katz technique). A questionnaire was completed to collect clinical-epidemiological data of interest. Results: In general, 5609 children were parasitized (29.5 %; 95 % CI: 28.9-30.2). The most frequent intestinal parasites were Blastocystis spp. (12.8 %; 95 % CI: 12.2-13.2) and Giardia duodenalis (8.6 %; 95 % CI: 8.2- 9.0). G. duodenalis was the most frequent parasite in eastern provinces and general giardiasis was significantly associated with a higher risk of infection in children who had contact with animals, ate unwashed fruits, drank untreated water, lived in rural residence zones, male sex, and had no hand washing practices after bathing. Investigation of associations between symptoms and Giardia infections revealed that diarrhea was statistically associated with children infected with this pathogen parasite (P < 0.05). Conclusions: Infections by G. duodenalis were the second most prevalent in Cuban children and it was associated with several risk factors. Diarrhea was associated with this infection in the studied children. The results obtained could be of great value for developing more effective health strategies to prevent and control giardiasis in children.

Neonatal calf diarrhoea (NCD) is a complex, multifactorial disease causing significant economic loss and impairing animal welfare worldwide. The most common infectious pathogens associated with NCD are Escherichia coli, bovine rotavirus, coronavirus, and Cryptosporidium parvum. It is commonly known that simultaneous infections (co-infections) with more than one pathogen occur during neonatal calf diarrhoea cases, but there is limited knowledge about how pathogens interact in co-infections and whether their combined effect results in greater pathology and clinical disease in neonatal calves. This study was performed to investigate whether co-infection with C. parvum and bovine coronavirus (BCoV) increases the severity and longevity of clinical disease and shedding of pathogens in neonatal calves compared to calves infected with either C. parvum or BCoV as single infection. Fifteen neonatal calves of different breeds and sex were used in the study. Calves were allocated into one of three infection groups on arrival at the research farm prior to oral inoculation of C. parvum and/or oral and intranasal inoculation of BCoV as follows Group 1 (Cp): inoculated with C. parvum; Group 2 (CpCo): Co-inoculated with C. parvum and BCoV, and Group 3 (Co): inoculated with BCoV. Animals (2-4 days of age) were inoculated sequentially, with C. parvum first and BCoV 48 hours later. Each calf was examined twice daily for clinical scores. Faecal samples were collected daily and analysed by (RT-)qPCR for quantification of pathogens. Cryptosporidium parvum subtypes were identified by sequencing PCR products from the gp60 gene. Faecal samples from 13/15 calves were positive by qPCR for C. parvum prior to experimental inoculation. Genotyping of faecal samples indicated natural and experimental cryptosporidiosis was caused by different genotypes of C. parvum. Calves inoculated with both C. parvum and BCoV (CpCo) and C. parvum (Cp) only, exhibited more clinical signs of a higher severity and over a longer period than calves inoculated with BCoV (Co) alone. Cp- and CpCo-calves had a higher frequency of liquid faeces than Co-calves. Both C. parvum and BCoV presented a similar shedding pattern in all groups throughout the experiment 

Investigations into the molecular epidemiology of Cryptosporidium have relied heavily on PCR-based genotyping, particularly at the small subunit rRNA (SSU rRNA) gene, to determine the species causing infection, and within-species subtyping by sequencing part of a highly polymorphic gene encoding a 60 kDa glycoprotein (gp60). The gp60 gene codes for an immunodominant surface glycoprotein that mediates host cell invasion and thus plays a role in host infectivity and pathogenicity, consistent with its highly polymorphic nature.  The sequence variations seen at this locus both within and between Cryptosporidium species provided features that are used to discriminate between isolates and the results incorporated in the nomenclature of the gp60 subtyping scheme. These features include sequence polymorphisms and mini- and microsatellite repeats. The gp60 subtyping scheme started with a simple set of nomenclature rules, involving a Roman numeral for the species designation (e.g. I for C. hominis, II for C. parvum), followed by a lower-case Latin letter to designate a gp60 family (e.g. Ia, Ib, IIa, etc), then the number of the TCA, TCG and TCT serine codons represented by (in turn) A, G and T (e.g. IeA11G3T3). Finally, rare “R” repeats present in only a few families each with a family-specific defined sequence (e.g. IIaA15G2R1, IfA12G1R5).  However, this nomenclature has evolved over the last 25 years as additional features have been discovered and described by different groups, such as new “r” repeats, “variants”, alphabetical suffixes, lack of serine repeat and, having exhausted Latin, use of the Greek alphabet. Some of these features differ depending on the species or allelic family. Thus, use of gp60 has become more complicated, and alternative interpretations have emerged, mostly due to the varied and confusing nature of the extended nomenclature, making it difficult to compare isolates.  In response to the evident need for standardisation, we have developed a comprehensive guide to standardise and promote a harmonised nomenclature for gp60 subtyping, including improved online resources and tools to aid the community in the use and future evolution of this scheme. In this poster, we will illustrate the various features underpinning a standardised nomenclature, highlighting pitfalls to avoid errors in interpretation, and signpost useful reference resources for those analysing this locus.  

Microaerophilic protistan infections contribute significant global disease burdens, disproportionately impacting developing countries and their public health systems. Three human microaerophilic protists, Giardia duodenalis, Trichomonas vaginalis and Entamoeba histolytica, infect ~400million people annually, causing gastrointestinal and urogenital tract infections and long-term, post-infectious sequalae. Treatments are limited to nitroheterocyclics; however, their reduced efficacy mean new drugs are urgently required. High-throughput screening (HTS) is key to identifying new candidates and compound classes with microaerophilic activity and new modes of action (MOA).  We used killing assays and performed HTS of a pure-compound microbial metabolite library containing a range of bioactive natural products.  We found 44 “hits” against T. vaginalis, 48 against T. foetus (a significant veterinary pathogen) and 74 against G. duodenalis at micromolar concentrations (<10 µM). With parallel counter-screens in cell lines, we show substantial overlaps between anti-protistal and anti-tumour activity, suggesting compounds impacting high metabolic demands of protists may have similar MOA as observed in tumours. We verified the nanomolar activity of a polyene anti-angiogenic with an active epoxide moiety. We modelled this compound’s activity in silico, confirming the molecular interactions of the active epoxide against putative targets in T. vaginalis, G. duodenalis and E. histolytica, defining a new rationale to refine epoxide-polyene candidates.  Overall, this contributes to the growing body of evidence which highlight the potential of microbial metabolites as novel anti-protistals.  

Giardiasis is a common parasitic infection caused by the protozoan Giardia lamblia, leading to gastrointestinal disturbances such as diarrhea, abdominal pain, and bloating. It is one of the most prevalent waterborne diseases worldwide, particularly affecting children in developed and developing countries. Despite the availability of treatments such as metronidazole, the increasing resistance of G. lamblia to conventional drugs and the side effects associated with long-term use have led to a search for alternative therapies. In this context, the therapeutic potential of plants from the Colombian Amazon has garnered significant interest due to their long history of use in folk medicine for treating various gastrointestinal disorders. This study evaluates the giardicidal activity of 15 crude hydroethanolic extracts from Amazonian plants against G. lamblia trophozoites. Using the MTT assay, the effectiveness of these extracts was tested at a concentration of 500 µg/mL against Giardia trophozoites (genotype A, WB/1267). Among the tested extracts, Attalea butyracea fruit extract (P-2) exhibited the strongest giardicidal activity, with a half-maximal inhibitory concentration (IC50) of 62.10 ± 6.57 µg/mL against the WB/1267 strain, and 100.90 ± 3.40 µg/mL against the GS/M strain (genotype B). Further analysis of P-2 revealed that at its IC50 concentration, it induced early apoptosis, while a significant increase in late apoptosis and necrosis was observed at 2xIC50. Immunofluorescence assay (IFA) and confocal microscopy confirmed chromatin condensation in treated trophozoites, and flow cytometry showed G1/S phase cell cycle arrest. Additionally, oxidative stress was induced, marked by increased reactive oxygen species (ROS), and structural damage was observed via IFA and transmission electron microscopy. Notably, Attalea butyracea extract synergized with metronidazole, the standard treatment for giardiasis. These findings highlight the potential of Colombian Amazon palm extracts, particularly Attalea butyracea, as promising candidates for further therapeutic development in the treatment of giardiasis.

Giardia duodenalis causes giardiasis in humans, companion, livestock and wild animals. Control of infection involves drugs as benzimidazoles (e.g., albendazole, ABZ) and 5-nitroheterocyclics [5-NHs: metronidazole (MTZ), furazolidone (FZD), nitazoxanide (NTZ)] as first-line agents. During infection, Giardia is exposed to immune and pro-oxidant host responses involving nitric oxide (NO). In Giardia, NO is detoxified by a flavohemoglobin (gFlHb), a heme-containing enzyme which is absent in mammals. gFlHb has NO dioxygenase and NADH oxidase activities converting NO into nitrate and producing a superoxide anion (O2•−) that causes oxidative stress and parasite death. The modulation of gFlHb activities may provide novel approaches for treatment of giardiasis. We investigated the capacity of selected benzimidazole-2-carbamates (BZCs: ABZ, oxibendazole, nocodazole), non-BZCs (thiabendazole), an ehtylphenylcarbamate (LQM-996) and 5-NHs (MTZ, NTZ, FZD and some derivatives) to bind to recombinant gFlHb at the heme group, modifying NADH consumption activity and/or inducing ROS production. Of these, BZCs and NTZ bind to heme and increased O2•− production (i.e. caused enzyme subversion), whereas MTZ binds to heme but inhibited NADH consumption. LQM-996 decreased NADH consumption and two out of four NTZ derivatives altered NADH oxidase activity. In silico docking and molecular dynamics studies suggested the interaction of distinct drug moieties in ABZ and NTZ with gFlHb sites involved in NADH and NO catalysis. These findings provide new insights on gFlHb as a novel target of BZCs, MTZ and NTZ, and provides a useful platform to assess the compounds binding capacity to gFlHb prior to experimental and clinical trials in giardiasis.

Giardia duodenalis is a widespread intestinal protozoan that affects mammals, including humans, causing symptoms such as abdominal pain and diarrhea. Treatment involves synthetic drugs like metronidazole (MTZ), but drug resistance is increasing. Thus, therapeutic alternatives are needed. Here we have examined the efficacy of the medicinal plant Tabebuia avellanedae dry extract (TD) and hydroalcoholic extract (TH), as well as one of its active compounds, beta-lapachone (β-lap), as potential treatment against G. duodenalis infection. In vitro activity (IC50 values after 48h) was quantified by viability assay on isolates of G. duodenalis Assemblage A and B, using MTZ as reference drug. In vitro cytotoxicity was evaluated on Caco-2 and MDCK cell lines and selectivity index (SI = IC50/CC50) determined at 48h post treatment. Furthermore, to better mimic intestinal conditions, drug efficacy as well as was cytotoxicity was assessed on infected versus non-infected intestinal Organoid Derived Monolayers (ODMs) in a transwell set-up by enumerating parasites 48 h after treatment and by measuring the transepithelial electrical resistance (TEER) and host cell viability. We observed good anti-G. duodenalis activity of all the compounds; β-lap demonstrated IC50 values lower than MTZ. The viability assays showed that TD exhibited toxicity at the highest concentration (2 mg/ml) after 12, 24, and 48 hours in both cell lines, whereas no cytotoxicity was observed for TH. Notably, the SI for TH approaches infinity as no cellular toxicity was observed. Despite very effective against G. duodenalis, β-lap proved to be toxic against both cell lines, likely due to its known anticancer activity (Gomes et al. 2021). In contrast, TD, TH and β-lap showed no significant cytotoxicity in ODMs, but still proofed effective against G. duodenalis in the ODM infection model, suggesting a suitable therapeutic window for intestinal application of T. avellanedae-derived antigiardial compounds. 

Parapipe is a modular, ISO-accreditable bioinformatics pipeline designed for high-throughput processing and analysis of Cryptosporidium NGS datasets, enabling high-resolution investigations of population structure, transmission dynamics, and genetic diversity. Parapipe integrates quality control, variant calling, whole-genome SNP typing, phylogenetics, and multiplicity of infection (MOI) detection into a streamlined workflow. Parapipe complements current methodological developments in DNA isolation and enrichment techniques, essential to generating data of sufficient quality to perform epidemiological analysis, by facilitating analysis and the generation of actionable data. Parapipe represents the first step in the development of a comprehensive suite of computational tools for gastrointestinal protozoan parasite genomic surveillance. This suite will include a relational database for storing genomic results and metadata, as well as a novel phylogenomic SNP neighborhood analysis tool for investigating SNP content and phylogenomic distance between stored samples. Together, these tools will provide an integrated platform for high-resolution epidemiological and evolutionary investigations. By integrating cutting-edge wet lab developments like baits capture with computational advancements, Parapipe represents a significant development in Cryptosporidium genomic surveillance. It establishes a scalable and reproducible framework for public health and research laboratories, enabling outbreak tracking, population diversity analysis, and the monitoring of emerging genotypes. Parapipe and the projected development of complementary tools represent a substantial development in genomic monitoring for cryptosporidiosis and other enteric protozoan diseases.

Colorectal cancer (CRC) is the third most common cancer globally and the second leading cause of cancer-related deaths. Some studies have shown that Cryptosporidium infection is significantly more frequent among CRC than non-CRC patients, suggesting that the parasite might have a role in tumor activity exacerbation and progression [1,2]. Similarly, a recent systematic review and meta-analysis study has shown that cancer patients were 1.24 times more likely to harbour G. duodenalis infection than healthy controls [3]. To ascertain the extent of these surveys, here we investigated the possible association of Cryptosporidium and/or Giardia infections with CRC development in a large, carefully recruited cohort of Colombian patients.  Individual stool samples were collected from 308 patients (age range: 45–69 years; male/female ratio: 0.7) undergoing CRC screening at five hospitals in Medellín, Colombia during the period September 2021 to June 2023. Specifically, patients having a diagnosis of colon or rectal cancer established by colonoscopy, computed tomography or magnetic resonance imaging and/or confirmed by anatomopathological analysis of tissue samples were included in the CRC group (n = 154). In parallel, patients with a normal colonoscopy were included in the non-CRC group (n = 154). To carry out the statistical analysis of the results under the best possible conditions, patients from each group were paired by sex and age. After genomic DNA extraction, detection and genotyping of Cryptosporidium and G. duodenalis was conducted by PCR and Sanger sequencing.  Cryptosporidium and G. duodenalis were detected in 0.3% (1/308; 95% CI: 0.01–1.8) and 7.5% (23/308; 95% CI: 4.8–11.0) of patients, respectively. No statistically significant differences were observed between the CRC and non-CRC groups regarding the presence of either Cryptospordium or Giardia infections. The Cryptosporidium-positive sample was identified as C. parvum but could only be characterised at the species level. The two Giardia-positive samples successfully genotyped were assigned to the assemblage B at the ssu rRNA marker. One of them was subsequently genotyped as sub-assemblage BIV at the gdh marker.  According to our data, there is no evidence supporting the hypothesis that Cryptosporidium and/or G. duodenalis infections predispose individuals to develop CRC.  

Giardia duodenalis is a species complex, which includes at least eight assemblages (A-H). These assemblages vary both in genetic structure and host specificity and multiple assemblages can be present in individual samples taken from a host or the environment. Yet, the prevalence of mixed-assemblage G. duodenalis infections and the presences of cysts from multiple assemblages are understudied. Our lab has developed a method that is able to detect G. duodenalis mixed-assemblage infections using next generation amplicon sequencing (NGS) of the beta-giardin gene. We have applied this method to studies in animal surveys and validated its use in combination with the US-FDA’s BAM Chapter 19b protocol for detection of G. duodenalis from fresh produce to ascertain the limit of detection of G. duodenalis on leafy greens. NGS outperforms traditional Sanger sequencing for the detection and differentiation of mixed infections in animal samples. The NGS method also provided robust detection of G. duodenalis on packaged leafy greens using the BAM Chapter 19B method coupled with assemblage-sensitive NGS. This method provides a new tool that can be used to explore mixed infections in prevalence studies and may also aid in outbreak investigations.

Giardia are highly divergent protists from an understudied eukaryotic supergroup. Giardia have a simple life cycle, cycling between the immotile but environmentally hardy cyst, and the flagellated trophozoite. During this process >50% of its genes are differentially transcribed. While in a host, trophozoites are challenged by host immunity and microbiome, as well as oxygen/food fluctuations and surviving in these complex environments must require robust and precise systems to regulate gene expression. Despite this, little is known about these systems in Giardia. Sequence specific Transcription Factors (TFs) are DNA binding proteins involved in gene expression regulation but are poorly described in even the best studied species Giardia lamblia, with just 31 TFs annotated in the reference genome (<0.5%), whereas we should expect ~3.5% based on the model organism Saccharomyces cerevisiae genome.

Here I will describe our efforts to reassess the number and types of TFs in Giardia, and to put this into context with other metamonads. By using a simple bioinformatic pipeline exploiting existing HMMs we have found additional unannotated TFs in the reference G. lamblia genome, as well as other members of the metamonad supergroup. This data show that Giardia is not unique in having relatively few TFs, and most metamonads had approximately 1% of their genome encoding canonical TFs. Interestingly another parasitic diplomonad, Spironucleus salmonicida, had approximately 2% of its genome encoding TFs, possibly due to a massive duplication of Myb-type HTH motif containing proteins. We found that irrespective of life-style (parasitic, symbiotic, or free-living) cells utilize a relatively similar number of canonical TFs, though all contain significantly less than model organisms. This may suggest that cryptic TF classes exist outside of opisthokonta or that alternative regulation methods are prevalent throughout relatively poorly described organisms e.g. Trypanosoma cruzi. Additionally we identified HTH motif to be the predominant canonical TF motif throughout the metamonad supergroup, whereas selected SAR organisms seem to prefer zinc-finger motifs, highlighting differences between supergroups.