Rapid visual detection of Giardia duodenalis in faecal samples using an RPA-CRISPR/Cas12a system.

Giardiasis is a common intestinal infection caused by Giardia duodenalis, which is a major economic and health burden for humans and livestock. Currently, a convenient and effective detection method is urgently needed. CRISPR/Cas12a-based diagnostic methods have been widely used for nucleic acid-based detection of pathogens due to their high efficiency and sensitivity. In this study, a technique combining CRISPR/Cas12a and RPA was established that allows the detection of G. duodenalis in faecal samples by the naked eye with high sensitivity (10-1 copies/µL) and specificity (no cross-reactivity with nine common pathogens). In clinical evaluations, the RPA-CRISPR/Cas12a-based detection assay detected Giardia positivity in 2% (1/50) of human faecal samples and 47% (33/70) of cattle faecal samples, respectively, which was consistent with the results of nested PCR. Our study demonstrated that the RPA-CRISPR/Cas12a technique for G. duodenalis is stable, efficient, sensitive, specific and has low equipment requirements. This technique offers new opportunities for on-site detection in remote and poor areas.

Giardia telomeres and telomerase.

Giardia duodenalis, the protozoan responsible for giardiasis, is a significant contributor to millions of diarrheal diseases worldwide. Despite the availability of treatments for this parasitic infection, therapeutic failures are alarmingly frequent. Thus, there is a clear need to identify new therapeutic targets. Giardia telomeres were previously identified, but our understanding of these structures and the critical role played by Giardia telomerase in maintaining genomic stability and its influence on cellular processes remains limited. In this regard, it is known that all Giardia chromosomes are capped by small telomeres, organized and protected by specific proteins that regulate their functions. To counteract natural telomere shortening and maintain high proliferation, Giardia exhibits constant telomerase activity and employs additional mechanisms, such as the formation of G-quadruplex structures and the involvement of transposable elements linked to telomeric repeats. Thus, this study aims to address the existing knowledge gap by compiling the available information (until 2023) about Giardia telomeres and telomerase, focusing on highlighting the distinctive features within this parasite. Furthermore, the potential feasibility of targeting Giardia telomeres and/or telomerase as an innovative therapeutic strategy is discussed.

Host-specific Cryptosporidium, Giardia and Enterocytozoon bieneusi in shelter dogs from central Europe.

Cryptosporidium spp., Giardia intestinalis and microsporidia are unicellular opportunistic pathogens that can cause gastrointestinal infections in both animals and humans. Since companion animals may serve as a source of infection, the aim of the present screening study was to analyse the prevalence of these intestinal protists in fecal samples collected from dogs living in 10 animal shelters in central Europe (101 dogs from Poland and 86 from the Czech Republic), combined with molecular subtyping of the detected organisms in order to assess their genetic diversity. Genus-specific polymerase chain reactions were performed to detect DNA of the tested species and to conduct molecular subtyping in collected samples, followed by statistical evaluation of the data obtained (using χ2 or Fisher's tests). The observed prevalence was 15.5, 10.2, 1 and 1% for G. intestinalis, Enterocytozoon bieneusi, Cryptosporidium spp. and Encephalitozoon cuniculi, respectively. Molecular evaluation has revealed the predominance of dog-specific genotypes (Cryptosporidium canis XXe1 subtype; G. intestinalis assemblages C and D; E. cuniculi genotype II; E. bieneusi genotypes D and PtEbIX), suggesting that shelter dogs do not pose a high risk of human transmission. Interestingly, the percentage distribution of the detected pathogens differed between both countries and individual shelters, suggesting that the risk of infection may be associated with conditions typical of a given location.

Zoonotic Cryptosporidium and Giardia in marsupials-an update.

Marsupials, inhabiting diverse ecosystems, including urban and peri-urban regions in Australasia and the Americas, intersect with human activities, leading to zoonotic spill-over and anthroponotic spill-back of pathogens, including Cryptosporidium and Giardia. This review assesses the current knowledge on the diversity of Cryptosporidium and Giardia species in marsupials, focusing on the potential zoonotic risks. Cryptosporidium fayeri and C. macropodum are the dominant species in marsupials, while in possums, the host-specific possum genotype dominates. Of these three species/genotypes, only C. fayeri has been identified in two humans and the zoonotic risk is considered low. Generally, oocyst shedding in marsupials is low, further supporting a low transmission risk. However, there is some evidence of spill-back of C. hominis into kangaroo populations, which requires continued monitoring. Although C. hominis does not appear to be established in small marsupials like possums, comprehensive screening and analysis are essential for a better understanding of the prevalence and potential establishment of zoonotic Cryptosporidium species in small marsupials. Both host-specific and zoonotic Giardia species have been identified in marsupials. The dominance of zoonotic G. duodenalis assemblages A and B in marsupials may result from spill-back from livestock and humans and it is not yet understood if these are transient or established infections. Future studies using multilocus typing tools and whole-genome sequencing are required for a better understanding of the zoonotic risk from Giardia infections in marsupials. Moreover, much more extensive screening of a wider range of marsupial species, particularly in peri-urban areas, is required to provide a clearer understanding of the zoonotic risk of Cryptosporidium and Giardia in marsupials.

Prevalence and assemblage of Giardia duodenalis in a case-control study of children under 5 years from Jimma, Southwest Ethiopia.

Giardia duodenalis is a common pathogenic intestinal protozoan parasite with high prevalence in developing countries, especially among children. The distribution of giardia assemblages among humans and their clinical relevance remains controversial. This study aimed to determine the prevalence and assemblage of Giardia among children under 5 years of age in Jimma, Southwest Ethiopia. Employing a case-control design, 606 children presenting with diarrhea at Jimma university medical center and Serbo Health Center were enrolled from December 2016 to July 2018 along with 617 matched controls without diarrhea. Giardia was detected and typed using real-time PCR. Univariate and multivariate regression analysis was performed. The total prevalence of Giardia was 41% (501/1223) and did not differ significantly between cases and controls (40% vs 42%). Prevalence increased by age, with the highest prevalence seen in children aged ≥ 25 months. Children without diarrhea with a history of diarrhea during the last month were more likely to be Giardia positive compared to children with no history diarrhea (OR 1.8 and 95%CI; 1.1-2.9). Regardless of current diarrhea symptoms, assemblage B predominated with 89%, followed by assemblage A (8%) and mixed infection assemblage A and B (3%). We report a high prevalence of Giardia by PCR detection in Jimma, Ethiopia, with assemblage B being predominant. There was a similar distribution of Giardia assemblages between children with and without diarrhea. Increasing age was a risk factor for Giardia infection. Community-based prevention and control strategies need to be employed to decrease the risk of giardia infection.

Epidemiology of giardiasis and assemblages A and B and effects on diarrhea and growth trajectories during the first 8 years of life: Analysis of a birth cohort in a rural district in tropical Ecuador.

BACKGROUND: There are limited longitudinal data on the acquisition of Giardia lamblia infections in childhood using molecular assays to detect and type assemblages, and measure effects of infections on diarrhea risk and childhood growth. METHODS: We analysed stool samples from a surveillance sample within a birth cohort in a rural district in tropical Ecuador. The cohort was followed to 8 years of age for the presence of G. lamblia in stools by quantitative PCR and A and B assemblages by Taqman assay or Sanger sequencing. We explored risk factors associated with infection using generalized estimating equations applied to longitudinal binary outcomes, and longitudinal panel data analysis to estimate effects of infection on diarrhea and growth trajectories. RESULTS: 2,812 stool samples collected between 1 month and 8 years of age from 498 children were analyzed and showed high rates of infection: 79.7% were infected at least once with peak prevalence (53.9%) at 5 years. Assemblage B was accounted for 56.8% of genotyped infections. Risk factors for infection included male sex (P = 0.001), daycare attendance (P<0.001), having a household latrine (P = 0.04), childhood (P<0.001) and maternal soil-transmitted helminth (P = 0.029) infections, and exposures to donkeys (age interaction P = 0.034). G. lamblia was associated with increased risk of diarrhea (per episode, RR 1.03, 95% CI 1.01-1.06, P = 0.011) during the first 3 years of life and a transient impairment of weight (age interaction P = 0.017) and height-for-age (age interaction P = 0.025) trajectories between 1 and 4 years of age. There was no increased risk of either assemblage being associated with outcomes. CONCLUSION: Our data show a relatively high edemicity of G. lamblia transmission during childhood in coastal Ecuador, and evidence that infection is associated with a transiently increased risk of diarrhea during the first 3 years of life and impairment of weight and height between 1 and 4 years.

Zoonotic Giardia duodenalis assemblage A in northern sloth from Brazilian Amazon.

BACKGROUND: The parasite Giardia duodenalis infects a wide range of vertebrate hosts, including domestic and wild animals as well as humans. Giardia is genotyped into eight assemblages (A-H). Zoonotic assemblages A and B have already been identified in humans and wild and domestic animals (non-human primates and cats) from Brazilian Amazon and in the world. Due to its zoonotic/zooanthroponotic nature, surveillance initiatives and the definition of Giardia assemblages are important in order to characterise the epidemiological scenario and to implement further control measures. OBJECTIVES: Determine assemblages of G. duodenalis in sloths from the Brazilian Amazon Region. METHODS: Faecal parasitological examination of sloths from Amazonas State. Polymerase chain reaction (PCR) targeting the beta giardin (BG), and genes from multilocus sequence typing (MLST) scheme, amplicon sequencing and phylogenetic analysis. FINDINGS: Here, we identified, by microscopy, Giardia in two northern sloths (Bradypus tridactylus). These two samples were submitted to molecular assays and it was revealed that both were infected by G. duodenalis assemblage A. Phylogenetic analysis showed that they belong to assemblage A within sequences from humans and wild and domestic animals. CONCLUSION: Therefore, besides showing, by the first time, the current presence of this parasite in sloths, our findings reveals that this wild animal species would be part of the zoonotic/zooanthroponotic scenario of this parasite in the Brazilian Amazon.

Efficacy of a membrane concentration method combined with real-time PCR for detection of Giardia and Cryptosporidium in drinking water.

The occurrence of Giardia and Cryptosporidium (oo)cysts in drinking source water poses a serious public health risk. Here, we established a method that combines membrane concentration and real-time polymerase chain reaction (PCR) to quantify Giardia and Cryptosporidium in drinking water. The water samples were filtered through a cellulose membrane to collect Giardia and Cryptosporidium, and then nucleic acids were extracted. Specific primers and probes were designed and synthesized according to the gph gene sequence of Giardia and 18S rRNA gene sequence of Cryptosporidium. The concentrations of the two targets were determined using real-time PCR technology. The sensitivity, specificity, and stability of the method were evaluated. Our findings revealed that the detection limits of real-time PCR method for detecting Giardia and Cryptosporidium were 0.926 and 0.65 copy/µL, respectively; the spiked recovery rates were above 60% and 38%, respectively, and relative standard deviations were under 0.95% and 2.26%, respectively. Therefore, this effective procedure based on the membrane concentration method and real-time PCR will be useful for detecting Giardia and Cryptosporidium in drinking water for purpose of continuous environmental monitoring.

Absence of Giardia spp. in fecal samples from capybaras (Hydrochoerus hydrochaeris) inhabiting urban areas in the state of Mato Grosso do Sul, Brazil.

Giardiasis is a major cause of diarrhea in humans and animals worldwide. Currently, there are nine species of Giardia, including Giardia duodenalis, which infects most vertebrates. The capybara (Hydrochoerus hydrochaeris) is the largest herbivorous rodent in the world. Although capybaras are hosts of several parasites of public health importance, including helminths and protozoa, there is a paucity of research on their zoonotic potential. We investigated the prevalence of Giardia spp. in populations of capybaras living in urban areas. Fecal samples from 247 capybaras were collected in Lagoa Maior, located in the municipality of Três Lagoas, and in Lago do Amor and Parque das Nações Indígenas, both located in the municipality of Campo Grande, state of Mato Grosso do Sul, Brazil. Fecal samples from capybaras originated from 133 adults (54%), 61 cubs (25%), and 53 juveniles (21%); 183 samples were collected in the rainy season and 64 in the dry season. Giardia spp. DNA was screened by the small-subunit ribosomal RNA (SSU rRNA) targeted PCR. Samples with DNA band sizes suggestive of Giardia spp. amplicons were examined by PCR targeting the glutamate dehydrogenase (GDH) and triosephosphate isomerase (TPI) genes. PCR amplicons were subjected to genetic sequencing. Nested PCR screening of the SSU rRNA gene revealed 16 samples showing faint DNA bands in gel electrophoresis with sizes similar to Giardia spp. amplicons. PCR amplicons of the SSU rRNA gene were analyzed by Sanger sequencing. Most of the sequencing reactions failed, and the chromatogram reads of some samples were ambiguous, suggesting nonspecific amplification. Therefore, all the capybara fecal samples were considered negative for Giardia spp. Two published studies on Giardia spp. in capybaras reported findings similar to ours, i.e., the absence or a low positivity rate for Giardia spp. However, further studies are needed to determine the possible role of capybaras in the epidemiology of giardiasis.

Fate of Cryptosporidium and Giardia through conventional and compact drinking water treatment plants.

Over the past three decades, a notable rise in the occurrence of enteric protozoan pathogens, especially Giardia and Cryptosporidium spp., in drinking water sources has been observed. This rise could be attributed not only to an actual increase in water contamination but also to improvements in detection methods. These waterborne pathogens have played a pivotal role in disease outbreaks and the overall escalation of disease rates in both developed and developing nations worldwide. Consequently, the control of waterborne diseases has become a vital component of public health policies and a primary objective of drinking water treatment plants (DWTPs). Limited studies applied real-time PCR (qPCR) and/or immunofluorescence assay (IFA) for monitoring Giardia and Cryptosporidium spp., particularly in developing countries like Egypt. Water samples from two conventional drinking water treatment plants and two compact units (CUs) were analyzed using both IFA and qPCR methods to detect Giardia and Cryptosporidium. Using qPCR and IFA, the conventional DWTPs showed complete removal of Giardia and Cryptosporidium, whereas Mansheyat Alqanater and Niklah CUs achieved only partial removal. Specifically, Cryptosporidium gene copies removal rates were 33.33% and 60% for Mansheyat Alqanater and Niklah CUs, respectively. Niklah CU also removed 50% of Giardia gene copies, but no Giardia gene copies were removed by Mansheyat Alqanater CU. Using IFA, both Mansheyat Alqanater and Niklah CUs showed a similar removal rate of 50% for Giardia cysts. Additionally, Niklah CU achieved a 50% removal of Cryptosporidium oocysts, whereas Mansheyat Alqanater CU did not show any removal of Cryptosporidium oocysts. Conventional DWTPs were more effective than CUs in removing enteric protozoa. The contamination of drinking water by enteric pathogenic protozoa remains a significant issue globally, leading to increased disease rates. Infectious disease surveillance in drinking water is an important epidemiological tool to monitor the health of a population.

Comparative study of a broad qPCR panel and centrifugal flotation for detection of gastrointestinal parasites in fecal samples from dogs and cats in the United States.

BACKGROUND: For decades, zinc sulfate centrifugal fecal flotation microscopy (ZCF) has been the mainstay technique for gastrointestinal (GI) parasite screening at veterinary clinics and laboratories. Elsewhere, PCR has replaced microscopy because of generally increased sensitivity and detection capabilities; however, until recently it has been unavailable commercially. Therefore, the primary aim of this study was to compare the performance of real-time PCR (qPCR) and ZCF for fecal parasite screening. Secondary aims included further characterization of markers for hookworm treatment resistance and Giardia spp. assemblages with zoonotic potential and qPCR optimization. METHODS: A convenience sampling of 931 canine/feline fecal samples submitted to a veterinary reference laboratory for routine ZCF from the Northeast US (11/2022) was subsequently evaluated by a broad qPCR panel following retention release. Detection frequency and agreement (kappa statistics) were evaluated between ZCF and qPCR for seven GI parasites [hookworm/(Ancylostoma spp.), roundworm/(Toxocara spp.), whipworm/(Trichuris spp.), Giardia duodenalis, Cystoisospora spp., Toxoplasma gondii, and Tritrichomonas blagburni] and detections per sample. Total detection frequencies were compared using a paired t-test; positive sample and co-infection frequencies were compared using Pearson's chi-squared test (p ≤ 0.05 significant) and qPCR frequency for hookworm benzimidazole (BZ) resistance (F167Y) and zoonotic Giardia spp. assemblage markers calculated. Confirmatory testing, characterization, and qPCR optimization were carried out with Sanger sequencing. RESULTS: qPCR detected a significantly higher overall parasite frequency (n = 679) compared to ZCF (n = 437) [p = < 0.0001, t = 14.38, degrees-of-freedom (df) = 930] and 2.6 × the co-infections [qPCR (n = 172) vs. ZCF (n = 66)], which was also significant (p = < 0.0001, X2 = 279.49; df = 1). While overall agreement of parasite detection was substantial [kappa = 0.74; (0.69-0.78], ZCF-undetected parasites reduced agreement for individual and co-infected samples. qPCR detected markers for Ancylostoma caninum BZ resistance (n = 5, 16.1%) and Giardia with zoonotic potential (n = 22, 9.1%) as well as two parasites undetected by ZCF (T. gondii/T. blagburni). Sanger sequencing detected novel roundworm species, and qPCR optimization provided detection beyond ZCF. CONCLUSIONS: These results demonstrate the statistically significant detection frequency advantage offered by qPCR compared to routine ZCF for both single and co-infections. While overall agreement was excellent, this rapid, commercially available qPCR panel offers benefits beyond ZCF with detection of markers for Giardia assemblages with zoonotic potential and hookworm (A. caninum) BZ resistance.

Metagenomic analysis reveals the relationship between intestinal protozoan parasites and the intestinal microecological balance in calves.

BACKGROUND: A close connection between a protozoan parasite and the balance of the other gut microbes of the host has been demonstrated. The calves may be naturally co-infected with many parasites, and the co-effects of parasites on other intestinal microbes of calves remain unclear. This study aims to preliminarily reveal the relationship between intestinal parasites and other intestinal microbes in calves. METHODS: Fecal samples were collected from four calves with bloody diarrhea, four calves with watery diarrhea, and seven normal calves, and the microbial flora of the samples were analyzed by whole-genome sequencing. Protozoal parasites were detected in the metagenome sequences and identified using polymerase chain reaction (PCR). RESULTS: Cryptosporidium, Eimeria, Giardia, Blastocystis, and Entamoeba were detected by metagenomic analysis, and the identified species were Giardia duodenalis assemblage E, Cryptosporidium bovis, Cryptosporidium ryanae, Eimeria bovis, Eimeria subspherica, Entamoeba bovis, and Blastocystis ST2 and ST10. Metagenomic analysis showed that the intestinal microbes of calves with diarrhea were disordered, especially in calves with bloody diarrhea. Furthermore, different parasites show distinct relationships with the intestinal microecology. Cryptosporidium, Eimeria, and Giardia were negatively correlated with various intestinal bacteria but positively correlated with some fungi. However, Blastocystis and Entamoeba were positively associated with other gut microbes. Twenty-seven biomarkers not only were significantly enriched in bloody diarrhea, watery diarrhea, and normal calves but were also associated with Eimeria, Cryptosporidium, and Giardia. Only Eimeria showed a distinct relationship with seven genera of bacteria, which were significantly enriched in the healthy calves. All 18 genera of fungi were positively correlated with Cryptosporidium, Eimeria, and Giardia, which were also significantly enriched in calves with bloody diarrhea. Functional genes related to parasites and diseases were found mainly in fungi. CONCLUSIONS: This study revealed the relationship between intestinal protozoan parasites and the other calf gut microbiome. Different intestinal protozoan parasites have diametrically opposite effects on other gut microecology, which not only affects bacteria in the gut, but also is significantly related to fungi and archaea.

Genetic diversity and molecular diagnosis of Giardia.

Giardia is a genus of flagellated protozoan parasites that infect the small intestine of humans and animals, causing the diarrheal illness known as giardiasis. Giardia exhibits significant genetic diversity among its isolates, which can have important implications for disease transmission and clinical presentation. This diversity is influenced by the coevolution of Giardia with its host, resulting in the development of unique genetic assemblages with distinct phenotypic characteristics. Although panmixia has not been observed, some assemblages appear to have a broader host range and exhibit higher transmission rates. Molecular diagnostic methods enable researchers to examine the genetic diversity of Giardia populations, enhancing our understanding of the genetic diversity, population structure, and transmission patterns of this pathogen and providing insights into clinical presentations of giardiasis.

The nucleolus of Giardia and its ribosomal biogenesis.

Giardia duodenalis is a protozoan intestinal parasite that causes a significant number of infections worldwide each year, particularly in low-income and developing countries. Despite the availability of treatments for this parasitic infection, treatment failures are alarmingly common. As a result, new therapeutic strategies are urgently needed to effectively combat this disease. On the other hand, within the eukaryotic nucleus, the nucleolus stands out as the most prominent structure. It plays a crucial role in coordinating ribosome biogenesis and is involved in vital processes such as maintaining genome stability, regulating cell cycle progression, controlling cell senescence, and responding to stress. Given its significance, the nucleolus presents itself as a valuable target for selectively inducing cell death in undesirable cells, making it a potential avenue for anti-Giardia treatments. Despite its potential importance, the Giardia nucleolus remains poorly studied and often overlooked. In light of this, the objective of this study is to provide a detailed molecular description of the structure and function of the Giardia nucleolus, with a primary focus on its involvement in ribosomal biogenesis. Likewise, it discusses the targeting of the Giardia nucleolus as a therapeutic strategy, its feasibility, and the challenges involved.

The Exosome-like Vesicles of Giardia Assemblages A, B, and E Are Involved in the Delivering of Distinct Small RNA from Parasite to Parasite.

The genetically related assemblages of the intestinal protozoa parasite Giardia lamblia are morphologically indistinguishable and are often derived from specific hosts. The Giardia assemblages are separated by large genetic distances, which might account for their relevant biological and pathogenic differences. In this work, we analyzed the RNAs cargo released into exosomal-like vesicles (ElVs) by the assemblages A and B, which differentially infect humans, and the assemblage E, which infects hoofed animals. The RNA sequencing analysis revealed that the ElVs of each assemblage contained distinct small RNA (sRNA) biotypes, suggesting a preference for specific packaging in each assemblage. These sRNAs were classified into three categories, ribosomal-small RNAs (rsRNAs), messenger-small RNAs (msRNAs), and transfer-small RNAs (tsRNAs), which may play a regulatory role in parasite communication and contribute to host-specificity and pathogenesis. Uptake experiments showed, for the first time, that ElVs were successfully internalized by the parasite trophozoites. Furthermore, we observed that the sRNAs contained inside these ElVs were first located below the plasma membrane but then distributed along the cytoplasm. Overall, the study provides new insights into the molecular mechanisms underlying the host-specificity and pathogenesis of G. lamblia and highlights the potential role of sRNAs in parasite communication and regulation.

Investigation of Giardia spp., Cryptosporidium spp. and Cyclospora cayetanensis in Samples Collected from Different Spring Waters Igdir, Türkiye

OBJECTIVE: In this study, it was aimed to investigate the physical and chemical properties of different spring waters and parasitic factors with different methods. METHODS: This study was carried out on 69 water samples collected from different spring waters in and around Igdir Province in April and June 2021. The samples were analyzed by native-Lugol, modified acid-fast staining, and nested polymerase chain reaction (nPCR). In addition, altitude (meter) and pressure (mmHg) measurements were made at the point where the water samples were taken. RESULTS: One or more parasites were detected in 27.5% of the 69 water samples examined. Only C. cayetanensis was found in 13% of the samples, only Cryptosporidium spp. in 10.1%, only Giardia spp. in 1.4%, only C. cayetanensis and Giardia spp. in 1.4%, only C. cayetanensis, Cryptosporidium spp., and Giardia spp. in 1.4%. Only Giardia spp. cyst (4.3%) was detected by the direct examination method. While C. cayetanensis and Cryptosporidium spp. oocysts were detected in 8.7% and 7.2% of the samples by the modified acid-fast staining method, C. cayetanensis was detected in 15.9% and Cryptosporidium spp. was detected in 11.6% of the samples by nPCR. When the C. cayetanensis and Cryptosporidium spp. positivity rates were compared according to the characteristics of the water, there was no statistical difference between the altitude, salinity, pH, mmHg, and temperature (kelvin) values, but a significant correlation was found between the amount of dissolved oxygen and Cryptosporidium spp. positivity (p=0.047). CONCLUSION: Cryptosporidium spp., C. cayetanensis, and G. intestinalis are important waterborne pathogens that can cause epidemics. It is our belief that in order to reduce the risk of contamination of these parasitic factors with spring waters, public awareness should be raised, infrastructures should be improved, and new water treatment techniques, such as ultraviolet, ozonation and monitoring systems, should be used.

A potential role for Giardia chaperone protein GdDnaJ in regulating Giardia proliferation and Giardiavirus replication.

BACKGROUND: Giardia duodenalis (referred to as Giardia) is a flagellated binucleate protozoan parasite, which causes one of the most common diarrheal diseases, giardiasis, worldwide. Giardia can be infected by Giardiavirus (GLV), a small endosymbiotic dsRNA virus belongs to the Totiviridae family. However, the regulation of GLV and a positive correlation between GLV and Giardia virulence is yet to be elucidated. METHODS: To identify potential regulators of GLV, we performed a yeast two-hybrid (Y2H) screen to search for interacting proteins of RdRp. GST pull-down, co-immunoprecipitation and bimolecular fluorescence complementation (BiFC) assay were used to verify the direct physical interaction between GLV RdRp and its new binding partner. In addition, their in vivo interaction and colocalization in Giardia trophozoites were examined by using Duolink proximal ligation assay (Duolink PLA). RESULTS: From Y2H screen, the Giardia chaperone protein, Giardia DnaJ (GdDnaJ), was identified as a new binding partner for GLV RdRp. The direct interaction between GdDnaJ and GLV RdRp was verified via GST pull-down, co-immunoprecipitation and BiFC. In addition, colocalization and in vivo interaction between GdDnaJ and RdRp in Giardia trophozoites were confirmed by Duolink PLA. Further analysis revealed that KNK437, the inhibitor of GdDnaJ, can significantly reduce the replication of GLVs and the proliferation of Giardia. CONCLUSION: Taken together, our results suggested a potential role of GdDnaJ in regulating Giardia proliferation and GLV replication through interaction with GLV RdRp.

Molecular characterization of Cryptosporidium and Giardia in environmental samples and faecal samples from biogas users in Bac Giang, Vietnam.

Little is known about Cryptosporidium and Giardia in biogas waste and humans in Vietnam. There is a potential risk of infections during or after using the biogas system. The detected protozoan genotypes are zoonotic pathogens, and contamination of vegetables may relay through runoff to the surface waters and soil. The objective of this study was to understand the role of the environment in the epidemiology of human infections in Bac Giang province, Vietnam, with a focus on investigating the presence of Cryptosporidium spp. genotypes and Giardia assemblages among 239 environmental samples and 94 faecal samples of biogas users. PCR and sequencing analysis were used to identify the occurrence and genotypes of Cryptosporidium and Giardia in these samples. Results showed that 13/333 (3.9 %) and 9/333 (2.7 %) samples were positive for Cryptosporidium oocyst and Giardia cysts, respectively. Characterization revealed the presence of Cryptosporidium scrofarum, C. suis, C. meleagridis, C. bailey and Giardia intestinalis assemblage A and E. C. scrofarum and Giardia assemblage E were identified for the first time in humans in Bac Giang. The current information from the above investigations will be valuable for protozoan source tracking and control interventions against Cryptosporidium and Giardia infection associated with biogas wastes in Vietnam.

Molecular characterization of waterborne protozoa in surface water and sediment in Brazil: a taxonomic survey of ciliated protozoa and their correlation with Giardia duodenalis and Cryptosporidium spp.

The detection of Giardia duodenalis and Cryptosporidium spp. was performed, along with the identification of the ciliated protozoa biodiversity, to evaluate the correlation between these protozoa in freshwater quality monitoring. Water and sediment samples from two sites in the Atibaia River (Campinas, São Paulo, Brazil) were collected monthly for 2 years (n = 96). Pathogenic protozoa in water and sediment were detected by using immunomagnetic separation, followed by visualization by immunofluorescence assay (IFA). All positive aliquots in IFA were subjected to DNA extraction and subsequently nested PCR. Qualitative (in vivo observation and silver impregnation) and quantitative (in vivo enumeration) analyses were performed for the ciliated protozoa. Giardia cysts were detected in 62.5% of the surface water samples and Cryptosporidium spp. in 25.0%. In the sediment, cysts were detected in 35.4% samples and oocysts in 16.6%. A total of 57 samples positive for Giardia cysts were subjected to sequencing, 40 of which were harboring G. duodenalis (24 were characterized as sub-assemblage AII). For ciliated protozoa, 73 taxa belonging to 53 genera were identified over the period of the study. These results revealed a high degree of contamination by waterborne protozoa in the main water source which supplies drinking water for more than one million people in Campinas (São Paulo), highlighting the need for continuous monitoring of this catchment site. In addition, the present study provides important data regarding the sources of the water body degradation, i.e., fecal contamination of human origin, in addition to the survey of the ciliated protozoa.

Highly contiguous genomes of human clinical isolates of Giardia duodenalis reveal assemblage- and sub-assemblage-specific presence-absence variation in protein-coding genes.

Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a widespread gastrointestinal protozoan parasite with debated taxonomic status. Currently, eight distinct genetic sub-groups, termed assemblages A-H, are defined based on a few genetic markers. Assemblages A and B may represent distinct species and are both of human public health relevance. Genomic studies are scarce and the few reference genomes available, in particular for assemblage B, are insufficient for adequate comparative genomics. Here, by combining long- and short-read sequences generated by PacBio and Illumina sequencing technologies, we provide nine annotated genome sequences for reference from new clinical isolates (four assemblage A and five assemblage B parasite isolates). Isolates chosen represent the currently accepted classification of sub-assemblages AI, AII, BIII and BIV. Synteny over the whole genome was generally high, but we report chromosome-level translocations as a feature that distinguishes assemblage A from B parasites. Orthologue gene group analysis was used to define gene content differences between assemblage A and B and to contribute a gene-set-based operational definition of respective taxonomic units. Giardia is tetraploid, and high allelic sequence heterogeneity (ASH) for assemblage B vs. assemblage A has been observed so far. Noteworthy, here we report an extremely low ASH (0.002%) for one of the assemblage B isolates (a value even lower than the reference assemblage A isolate WB-C6). This challenges the view of low ASH being a notable feature that distinguishes assemblage A from B parasites, and low ASH allowed assembly of the most contiguous assemblage B genome currently available for reference. In conclusion, the description of nine highly contiguous genome assemblies of new isolates of G. duodenalis assemblage A and B adds to our understanding of the genomics and species population structure of this widespread zoonotic parasite.