Establishing the Performance of Next-Generation Amplicon Sequencing for Detection of Giardia duodenalis in Ready-to-Eat Packaged Leafy Greens.

Giardia duodenalis is a globally distributed intestinal parasite that commonly infects both humans and animals. G. duodenalis is a species complex, which includes eight assemblages that vary both in genetic structure and host specificity. The prevalence of mixed-assemblage G. duodenalis cysts on food, an understudied infection route for G. duodenalis, remains unknown. In the present study, a method able to detect G. duodenalis mixed-assemblage infections using next-generation amplicon sequencing (NGS) of the beta-giardin gene was applied in combination with the US-FDA's BAM Chapter 19b protocol for the detection of G. duodenalis from fresh produce to ascertain the limit of detection of G. duodenalis on leafy greens. Ready-to-eat baby Romaine lettuce was inoculated with 5 (n = 5), 20 (n = 10), 100 (n = 10), 200 (n = 10), or 1,000 (n = 10) G. duodenalis cysts of the assemblage B strain H3. Detection of G. duodenalis was successful in 100% of the samples seeded with 1,000, 200, and 100 cysts, in 50% of the samples seeded with 20 cysts, and in none of the samples seeded with 5 cysts. We thus demonstrate robust detection of G. duodenalis on packaged leafy greens using the BAM Chapter 19B method coupled with assemblage-sensitive NGS. This protocol provides a new diagnostic tool useful for both prevalence studies and outbreak investigations involving fresh produce that may assist in better describing the role of G. duodenalis in foodborne illness and in protecting consumers from contaminated fresh produce.

Lysine methyltransferase 2 plays a key role in the encystation process in the parasite Giardia lamblia.

Histone post-translational modifications are extensively studied for their role in regulating gene transcription and cellular environmental adaptation. Research into these modifications has recently begun in the protozoan parasite Giardia lamblia, focusing on histone-modifying enzymes and specific post-translational changes. In the transformation from the trophozoite to the cyst form in the life cycle of this parasite, significant morphological and genetic alterations occur, culminating in the synthesis of cyst wall proteins responsible for forming the protective cyst wall. It has been previously demonstrated that histone deacetylation is required during encystation and that the enzyme lysine methyltransferase 1 is involved in the upregulation of encystation. Our study aims to extend the analysis to lysine methyltransferase 2 (GlKMT2) function. For this, two constructs were generated: one that downregulate the expression of GLKMT2 via antisense (glkmt2-as transgenic cells) and the other overexpressing GlKMT2 (glkmt2-ha transgenic cells). We found that the glktm2-as transgenic cells showed an arrest in progress at the late encystation stage. Consequently, the number of cysts produced was lower than that of the control cells. On the other hand, we found that the overexpression of GlKMT2 acts as a negative mutant of the enzyme. In this way, these glktm2-ha transgenic cells showed the same behavior during growth and encystation as glkmt2-as transgenic cells. This interplay between different enzymes acting during encystation reveals the complex process behind the differentiation of the parasite. Understanding how these enzymes play their role during the encystation of the parasite would allow the design of inhibitors to control the parasite.

Comparative genomics of Giardia duodenalis sub-assemblage AI beaver (Be-2) and human (WB-C6) strains show remarkable homozygosity, sequence similarity, and conservation of VSP genes.

Giardia duodenalis, a major cause of waterborne infection, infects a wide range of mammalian hosts and is subdivided into eight genetically well-defined assemblages named A through H. However, fragmented genomes and a lack of comparative analysis within and between the assemblages render unclear the molecular mechanisms controlling host specificity and differential disease outcomes. To address this, we generated a near-complete de novo genome of AI assemblage using the Oxford Nanopore platform by sequencing the Be-2 genome. We generated 148,144 long-reads with quality scores of > 7. The final genome assembly consists of only nine contigs with an N50 of 3,045,186 bp. This assembly agrees closely with the assembly of another strain in the AI assemblage (WB-C6). However, a critical difference is that a region previously placed in the five-prime region of Chr5 belongs to Chr4 of Be-2. We find a high degree of conservation in the ploidy, homozygosity, and the presence of cysteine-rich variant-specific surface proteins (VSPs) within the AI assemblage. Our assembly provides a nearly complete genome of a member of the AI assemblage of G. duodenalis, aiding population genomic studies capable of elucidating Giardia transmission, host range, and pathogenicity.

Exploration of Giardia small nucleolar RNAs (snoRNAs) and their possible microRNA derivatives.

Small nucleolar RNAs (snoRNAs) are short non-coding RNAs that are abundant in the nucleoli of eukaryotic cells and play a crucial role in various aspects of ribosomal RNA (rRNA) maturation, including modifications such as 2'-O-methylation or pseudouridylation. On the other hand, Giardia duodenalis is a microaerophilic, flagellated, binucleate protozoan responsible for causing giardiasis. Although numerous snoRNAs have been detected in Giardia, their investigation remains limited. Nevertheless, they have been found to play a crucial role in the rRNA precursor processing pathway and influence other cellular functions. In addition, it has been proposed that some microRNAs are generated from these snoRNAs through excision by the Giardia endoribonuclease Dicer. These microRNAs are believed to contribute to the regulation of antigenic variation, which allows the parasite to evade the host immune response. Specifically, they play a role in modulating variant-specific surface proteins (VSPs) and other cysteine-rich surface antigens (CSAs). The main objective of this study was to bring together the available data on snoRNAs in Giardia, uncovering their functions in various processes and their importance on a global scale. In addition, the research delved into potential microRNAs speculated to originate from snoRNAs, exploring their impact on cellular processes.

Multiplex PCR for bacterial, viral and protozoal pathogens in persistent diarrhoea or persistent abdominal pain in Côte d'Ivoire, Mali and Nepal.

In contrast to acute diarrhoea, the aetiology of persistent digestive disorders (≥ 14 days) is poorly understood in low-resource settings and conventional diagnostic approaches lack accuracy. In this multi-country study, we compared multiplex real-time PCR for enteric bacterial, parasitic and viral pathogens in stool samples from symptomatic patients and matched asymptomatic controls in Côte d'Ivoire, Mali and Nepal. Among 1826 stool samples, the prevalence of most pathogens was highest in Mali, being up to threefold higher than in Côte d'Ivoire and up to tenfold higher than in Nepal. In all settings, the most prevalent bacteria were EAEC (13.0-39.9%) and Campylobacter spp. (3.9-35.3%). Giardia intestinalis was the predominant intestinal protozoon (2.9-20.5%), and adenovirus 40/41 was the most frequently observed viral pathogen (6.3-25.1%). Significantly different prevalences between symptomatic and asymptomatic individuals were observed for Campylobacter, EIEC and ETEC in the two African sites, and for norovirus in Nepal. Multiple species pathogen infection was common in Côte d'Ivoire and Mali, but rarely found in Nepal. We observed that molecular testing detected multiple enteric pathogens and showed low discriminatory accuracy to distinguish between symptomatic and asymptomatic individuals. Yet, multiplex PCR allowed for direct comparison between different countries and revealed considerable setting-specificity.

Comparative performance evaluation of four different methods for diagnosing Giardia infection in dogs and zoonotic assemblages' identification.

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

Giardia intestinalis extracellular vesicles induce changes in gene expression in human intestinal epithelial cells in vitro.

Giardiasis is a common waterborne zoonotic disease caused by Giardia intestinalis. Upon infection, Giardia releases excretory and secretory products (ESPs) including secreted proteins (SPs) and extracellular vesicles (EVs). Although the interplay between ESPs and intestinal epithelial cells (IECs) has been previously described, the functions of EVs in these interactions and their differences from those of SPs require further exploration. In the present study, EVs and EV-depleted SPs were isolated from Giardia ESPs. Proteomic analyses of isolated SPs and EVs showed 146 and 91 proteins, respectively. Certain unique and enriched proteins have been identified in SPs and EVs. Transcriptome analysis of Caco-2 cells exposed to EVs showed 96 differentially expressed genes (DEGs), with 56 upregulated and 40 downregulated genes. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) indicated that Caco-2 genes related to metabolic processes, the HIF-1 signaling pathway, and the cAMP signaling pathway were affected. This study provides new insights into host-parasite interactions, highlighting the potential significance of EVs on IECs during infections.

Modeling Studies of the Mechanism of Context-Dependent Bidirectional Movements of Kinesin-14 Motors.

Kinesin-14s, a subfamily of the large superfamily of kinesin motor proteins, function mainly in spindle assembly and maintenance during mitosis and meiosis. KlpA from Aspergillus nidulans and GiKIN14a from Giardia intestinalis are two types of kinesin-14s. Available experimental results puzzlingly showed that while KlpA moves preferentially toward the minus end in microtubule-gliding setups and inside parallel microtubule overlaps, it moves preferentially toward the plus end on single microtubules. More puzzlingly, the insertion of an extra polypeptide linker in the central region of the neck stalk switches the motility direction of KlpA on single microtubules to the minus end. Prior experimental results showed that GiKIN14a moves preferentially toward the minus end on single microtubules in either tailless or full-length forms. The tail not only greatly enhances the processivity but also accelerates the ATPase rate and velocity of GiKIN14a. The insertion of an extra polypeptide linker in the central region of the neck stalk reduces the ATPase rate of GiKIN14a. However, the underlying mechanism of these puzzling dynamical features for KlpA and GiKIN14a is unclear. Here, to understand this mechanism, the dynamics of KlpA and GiKIN14a were studied theoretically on the basis of the proposed model, incorporating potential changes between the kinesin head and microtubule, as well as the potential between the tail and microtubule. The theoretical results quantitatively explain the available experimental results and provide predicted results. It was found that the elasticity of the neck stalk determines the directionality of KlpA on single microtubules and affects the ATPase rate and velocity of GiKIN14a on single microtubules.

Microscopy detection and molecular characterisation of Giardia duodenalis infection in outpatients seeking medical care in Egypt.

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

Development of New PCR Protocols to Detect Genetic Diversity in the Metronidazole Metabolism Genes in Susceptible and Refractory Clinical Samples of Giardia duodenalis.

PURPOSE: Investigating the genetic variation in thioredoxin reductase (TrxR) and nitroreductase (NR) genes in both treatment-resistant and -sensitive Giardia duodenalis isolates can provide valuable information in identifying potential markers of resistance to metronidazole. The rapid increase in metronidazole treatment failures suggests the presence of genetic resistance mechanisms. By analyzing these genes, researchers can gain insights into the efficacy of metronidazole against G. duodenalis and potentially develop alternative treatment strategies. In this regard, four G. duodenalis isolates (two clinically sensitive and two clinically resistant to metronidazole) were collected from various hospitals of Shiraz, southwestern Iran. METHODS: Parasitological methods including sucrose flotation and microscopy were employed for the primary confirmation of G. duodenalis cysts in stool samples. Microscopy-positive samples were approved by SSU-PCR amplification of the parasite DNA. All four positive G. duodenalis specimens at SSU-PCR were afterward analyzed utilizing designed primers based on important metronidazole metabolism genes including TrxR, NR1, and NR2. RESULTS: Unlike TrxR gene, the results of NR1 and NR2 genes showed that there are non-synonymous variations between sequences of treatment-sensitive and -resistant samples compared to reference sequences. Furthermore, the outcomes of molecular docking revealed that there is an interaction between the protein sequence and spatial shape of treatment-resistant samples and metronidazole in the position of serine amino acid based on the NR1 gene. CONCLUSION: This issue can be one of the possible factors involved in the resistance of Giardia parasites to metronidazole. To reach more accurate results, a large sample size along with simulation and advanced molecular dynamics investigations are needed.

Detection of zoonotic enteropathogens in captive large felids in Italy.

AIMS: Within the One Health paradigm, infectious disease surveillance have been developed for domestic and wild animals, leaving the role of captive non-domestic populations, especially felids in zoos and circuses, less explored. This study addresses the proximity of these captive animals to urban areas, necessitating focused monitoring for potential zoonotic enteropathogens. The present work aimed to investigate the presence of such zoonotic enteropathogens in faecal samples from captive large felid populations. METHODS AND RESULTS: A total of 108 faecal samples were collected in three circuses, five zoos and one rescue centre across Italy. Salmonella spp. isolation, serotyping and antimicrobial susceptibility testing were conducted on all samples. Additionally, 60 samples were also examined for gastrointestinal parasites using standard coprological techniques. Giardia spp. detection employed direct immunofluorescent staining and specific PCR, while Toxoplasma gondii was detected using PCR targeting B1 gene. A total of 51 Salmonella enterica subsp. enterica were isolated, with predominant serovariants including Infantis (43.1%), Coeln (11.8%) and Newport (11.8%). The captive felids likely act as asymptomatic carriers of foodborne Salmonella, with notable resistance ampicillin and trimethoprim-sulfamethoxazole, no resistance to enrofloxacin was noted. Microscopic analysis revealed Toxascaris leonina eggs in 11 faecal samples (18.3%) and Giardia duodenalis cysts in one animal (1.7%). CONCLUSIONS: Captive animals in public settings may act as sources of Salmonella infection and enteroparasitosis for both occupational and general exposure. The study emphasizes the role of captive animals in antimicrobial resistance dynamics, highlighting the need for routine pathogen screening in the management practices of zoological structures.

The detection and phylogenetic characterization of Cryptosporidium, Cystoisospora, and Giardia duodenalis of cats in South Korea.

Introduction: Cryptosporidium, Cystoisospora, and Giardia duodenalis are gastrointestinal protozoa parasites that cause diarrhea in various animals. However, information regarding the detection and phylogenetic characterization of gastrointestinal protozoa parasites in cats is limited throughout South Korea. Therefore, this study aimed to determine the detection and identify subspecies of gastrointestinal protozoa parasites in cats from South Korea. Methods: A total of 290 fecal samples were collected from stray, companion, and shelter cats in six provinces. Cryptosporidium, Cystoisospora, and G. duodenalis were identified by PCR. All positive samples were subtyped by PCR and sequencing of gp60, ITS-1, tpi, bg, and gdh. Results: The overall detection of gastrointestinal protozoan parasitic infection was 17.93%. G. duodenalis was the most prevalent, with 7.93%, followed by Cystoisospora spp. (7.24%) and Cryptosporidium spp. (4.48%). In addition, C. felis (n=10), C. parvum (n=2), C. ryanae (n=1), Cystoisospora felis (n=14), Cystoisospora suis (n=5), Cystoisospora ohioensis (n=1), Cystoisospora spp. were identified in subspecies analysis of positive samples. C. felis showed a significant association with diarrhea (7.81%) and living condition (6.04%), and Cystoisospora felis in diarreha (9.38%) according to detection. Through phylogenetic analysis of the tpi, bg, and gdh genes from 23 G. duodenalispositive samples, it was confirmed that the samples of present study belonged to assemblage A, B, C, and D. Discussion: South Korean cats have a high rate of gastrointestinal protozoan parasites infection with cat-specific Cryptosporidium and Cystoisospora, which are associated with living conditions and diarrhea symptoms. Moreover, zoonotic and other animal-specific subtype of protozoan parasites have been detected in cat feces.

Giardia VSPAS7 protein attenuates Giardia intestinalis-induced host macrophage pyroptosis.

BACKGROUND: The unicellular protozoan parasite Giardia intestinalis, which primarily infects humans and animals such as cattle and sheep, is having a major negative impact on public health. Giardia is able to evade the recognition and elimination of the host immune system because of the trophozoite surface and extracellular vesicles (EVs) covered by variant-specific surface proteins (VSPs). As key proteins for immune evasion, whether VSPs can regulate Giardia-induced pyroptosis and promote Giardia evasion of host immune responses has not been reported. METHODS: To examine the role of Giardia VSPAS7 on Giardia-induced activation of the signaling pathway, secretion of pro-inflammatory cytokines, pyroptosis and the mechanism involved, we constructed the pcDNA3.1-vspas7 expression plasmid and transfected this plasmid into mouse macrophages. Key proteins for pyroptosis, IL-1ß secretion and LDH release were detected in pcDNA3.1-vspas7-transfected wild-type (WT) cells and NLRP3-deficient cells by western blot, ELISA and LDH assays, respectively. The interactions of Giardia VSPAS7 and mouse NLRP3 were examined using immunofluorescence assays (IFA), co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays. RESULTS: VSPAS7 could decrease the levels of phosphorylated-p65 (P-p65), P-IκBα and P-ERK caused by Giardia and reduce the production levels of Giardia-induced pro-inflammatory cytokine IL-6, IL-12 p40 and TNF-α. The results showed that VSPAS7 inhibited Giardia-mediated activation of NF-κB, ERK/MAPK signaling and secretion of pro-inflammatory cytokines. Furthermore, VSPAS7 suppressed Giardia-induced macrophage pyroptosis by reducing GSDMD cleavage, caspase-1 activation, IL-1ß secretion and LDH release. We further found that VSPAS7 could interact with mouse NLRP3 directly, and in NLRP3-deficient cells the suppression of Giardia-induced macrophage pyroptosis by VSPAS7 was significantly attenuated. CONCLUSIONS: Overall, VSPAS7 could inhibit Giardia-induced activation of signaling pathways and pyroptosis in host macrophages, allowing Giardia evasion of host immune responses. Studies on Giardia VSP-mediated immune evasion provide an important theoretical basis for in-depth studies on Giardia pathogenicity.

Prevalence and public health relevance of enteric parasites in domestic dogs and cats in the region of Madrid (Spain) with an emphasis on Giardia duodenalis and Cryptosporidium sp.

BACKGROUND: Pet dogs and cats exert an unquestionable beneficial effect in the well-being of their owners, but can also act as a source of zoonotic infections if improperly cared. OBJECTIVES: We investigated the occurrence, risk factors, genetic variability and zoonotic potential of intestinal parasites in dogs and cats attended in a clinical veterinary setting in Spain. METHODS: Canine (n = 252) and feline (n = 35) faecal samples were collected during 2017-2019 and analysed by coproparasitological methods. A rapid lateral immunochromatographic test (ICT) was used for detecting Giardia duodenalis and Cryptosporidium sp. Samples positive at microscopy examination and/or ICT were reassessed by molecular methods. RESULTS: Overall, 48.8% (123/252) of dogs and 48.6% (17/35) of cats were infected by enteric parasites. In dogs, G. duodenalis was the most prevalent species (40.9%), followed by Cystoisospora sp. (7.1%), and Toxocara canis (5.2%). In cats, Joyeuxiella sp. and Toxocara cati were the dominant species (20.0% each), followed by G. duodenalis (14.3%), D. caninum (5.7%) and Cystoisospora felis and Toxascaris leonina (2.9% each). Pups and kittens were more likely to harbour intestinal parasites and develop clinical signs. Sequence analyses of dog isolates revealed the presence of assemblages A (n = 1), C (n = 4), D (n = 4) and C+D (n = 1) within G. duodenalis; C. parvum (n = 1) and C. canis (n = 4) within Cryptosporidium and PtEb IX (n = 1) in Enterocytozoon bieneusi. A novel C. canis subtype family, named XXi, is reported. CONCLUSIONS: Our results highlight that (i) well-cared dogs carry zoonotic enteric protozoan parasites of public health relevance, (ii) proper hygiene practices and routine veterinary treatment are essential to prevent zoonotic infections, (iii) vulnerable populations should avoid contact with pups/kittens with diarrhoea and (iv) infected dogs might be major contributors to the environmental contamination with soil-transmitted helminths (STHs) eggs.

Occurrence and limited zoonotic potential of Cryptosporidium spp., Giardia duodenalis, and Balantioides coli infections in free-ranging and farmed wild ungulates in Spain.

Little information is currently available on the occurrence and molecular diversity of the enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis, and Balantioides coli in wild ungulates and the role of these host species as potential sources of environmental contamination and consequent human infections. The presence of these three pathogens was investigated in eight wild ungulate species present in Spain (genera Ammotragus, Capra, Capreolus, Cervus, Dama, Ovis, Rupicapra, and Sus) by molecular methods. Faecal samples were retrospectively collected from free-ranging (n = 1058) and farmed (n = 324) wild ungulates from the five Spanish bioregions. Overall infection rates were 3.0% (42/1382; 95% CI: 2.1-3.9%) for Cryptosporidium spp., 5.4% (74/1382; 95% CI: 4.2-6.5%) for G. duodenalis, and 0.7% (9/1382; 95% CI: 0.3-1.2%) for B. coli. Cryptosporidium infection was detected in roe deer (7.5%), wild boar (7.0%) and red deer (1.5%), and G. duodenalis in southern chamois (12.9%), mouflon (10.0%), Iberian wild goat (9.0%), roe deer (7.5%), wild boar (5.6%), fallow deer (5.2%) and red deer (3.8%). Balantioides coli was only detected in wild boar (2.5%, 9/359). Sequence analyses revealed the presence of six distinct Cryptosporidium species: C. ryanae in red deer, roe deer, and wild boar; C. parvum in red deer and wild boar; C. ubiquitum in roe deer; C. scrofarum in wild boar; C. canis in roe deer; and C. suis in red deer. Zoonotic assemblages A and B were detected in wild boar and red deer, respectively. Ungulate-adapted assemblage E was identified in mouflon, red deer, and southern chamois. Attempts to genotype samples positive for B. coli failed. Sporadic infections by canine- or swine-adapted species may be indicative of potential cross-species transmission, although spurious infections cannot be ruled out. Molecular evidence gathered is consistent with parasite mild infections and limited environmental contamination with (oo)cysts. Free-ranging wild ungulate species would not presumably play a significant role as source of human infections by these pathogens. Wild ruminants do not seem to be susceptible hosts for B. coli.

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.

Molecular identification and genetic characteristics of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in human immunodeficiency virus/acquired immunodeficiency syndrome patients in Shanghai, China.

BACKGROUND: Opportunistic infections are a ubiquitous complication in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients. Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are common opportunistic intestinal pathogens in humans. In China, despite the number of HIV/AIDS patients being extremely large, only a few studies have investigated opportunistic infections caused by intestinal pathogens in this patient population. The aims of this study were to elucidate the occurrence and genetic characteristics of Cryptosporidium spp., G. duodenalis, and E. bieneusi in HIV/AIDS patients. METHODS: We collected fecal specimens from 155 HIV/AIDS patients (one from each patient). All of the specimens were examined for the presence of the pathogens by genotyping using polymerase chain reaction and sequencing of the small subunit ribosomal RNA gene for Cryptosporidium spp.; the triosephosphate isomerase, ß-giardin and glutamate dehydrogenase genes for G. duodenalis; and the internal transcribed spacer region of the rRNA gene for E. bieneusi. The Cryptosporidium-positive specimens were further subtyped by polymerase chain reacion and sequencing of the 60-kDa glycoprotein gene. RESULTS: Six (3.9%), three (1.9%), and eight (5.2%) HIV/AIDS patients were positive for Cryptosporidium spp., G. duodenalis, and E. bieneusi, respectively. No statistical differences were observed in occurrence rate between the groups by gender, clinical symptom (diarrhea), and CD4+ cell count. Four Cryptosporidium species were identified: Cryptosporidium hominis (n = 2), Cryptosporidium parvum (n = 1), Cryptosporidium meleagridis (n = 1), and Cryptosporidium andersoni (n = 2). Furthermore, two C. hominis subtypes (IeA12G3T3 and IaA28R4) were detected. Three G. duodenalis-positive specimens were successfully amplified and sequenced at the triosephosphate isomerase and ß-giardin loci, which led to the identification of assemblages C and B, respectively. Seven genotypes (D, Type IV, EbpC, Peru11, EbpD, A, and I) were identified in E. bieneusi-positive specimens. CONCLUSIONS: Our findings should increase awareness of AIDS-related opportunistic intestinal pathogens, and indicate the need for routine examination in clinical practice for the detection of Cryptosporidium spp., G. duodenalis, and E. bieneusi. Homology analyses of the three intestinal pathogens at the nucleotide and/or amino acid levels indicated their zoonotic potential.

Gastrointestinal parasites in young dogs and risk factors associated with infection.

Young dogs are particularly susceptible to infections with endoparasites. The occurrence of endoparasites was investigated in young dogs from Central Germany between July 2020 and July 2022. In total, 386 fecal samples originating from 171 dogs were examined for the prevalence of endoparasites using a combined flotation- and sedimentation technique and conventional PCR. Overall, in 41.2% (159/386) of the examined samples, endoparasites were detected. The most frequently occurring endoparasites were Giardia duodenalis (29%), Cryptosporidium spp. (9.1%), Cystoisospora spp. (7.3%), and Toxocara canis (6%). Sequencing of G. duodenalis positive samples showed that most infections belonged to the host-specific assemblages C (38.4% (43/112)) and D (35.7% (40/112)). The zoonotic assemblage A was identified in 8% (9/112) of the samples. Moreover, mixed infections were observed as follows: C/D in 5 (4.5%), D/A in 4 (3.6%), and C/A in 3 (2.7%) samples. All assemblage A infections were assigned to the potentially zoonotic subassemblage AI. Co-infections of G. duodenalis and Cryptosporidium spp. were observed in 3.1% (12/386) of the samples. Analyzing several host factors for their potential association with endoparasitic infection, the origin of dogs, as well as the living environment were identified as the main risk factors for infection with endoparasites. Overall, this study shows a high infection rate with endoparasites, especially G. duodenalis, in young dogs from Germany. The results of this study contribute to further insight into the distribution and potential risk factors associated with endoparasitic infections, as well as the zoonotic potential these parasites may present.

Identification of target genes regulated by encystation-induced transcription factor Myb2 using knockout mutagenesis in Giardia lamblia.

BACKGROUND: Encystation is one of the two processes comprising the life cycle of Giardia lamblia, a protozoan pathogen with tetraploid genome. Giardia lamblia Myb2 (GlMyb2) is a distinct encystation-induced transcription factor whose binding sites are found in the promoter regions of many encystation-induced genes, including its own. METHODS: Two sequential CRISPR/Cas9 experiments were performed to remove four glmyb2 alleles. The expression level of G. lamblia cyst wall protein 1 (GlCWP1), a well-known target gene of GlMyb2, was measured via western blotting and immunofluorescence assays. Chromatin immunoprecipitation experiments using anti-GlMyb2 antibodies were performed on the encysting G. lamblia cells. Quantitative real-time PCR was performed to confirm an expression of candidate GlMyb2-regulated genes by comparing the transcript level for each target candidate in wild-type and knockout mutant Giardia. The promoter region of glcwp1 was analyzed via deletion and point mutagenesis of the putative GlMyb2 binding sites in luciferase reporters. RESULTS: Characterization of the null glmyb2 mutant indicated loss of functions related to encystation, i.e. cyst formation, and expression of GlCWP1. The addition of the wild-type glmyb2 gene to the null mutant restored the defects in encystation. Chromatin immunoprecipitation experiments revealed dozens of target genes. Nineteen genes were confirmed as GlMyb2 regulons, which include the glmyb2 gene, six for cyst wall proteins, five for signal transduction, two for transporter, two for metabolic enzymes, and three with unknown functions. Detailed analysis on the promoter region of glcwp1 defined three GlMyb2 binding sites important in its encystation-induced expression. CONCLUSIONS: Our data confirm that GlMyb2 acts as a transcription activator especially during encystation by comparing the glmyb2 knockout mutant with the wild type. Further investigation using glmyb2 null mutant will provide knowledge regarding transcriptional apparatus required for the encystation process of G. lamblia.

Lack of efficacy of fenbendazole against Giardia duodenalis in a naturally infected population of dogs in France.

Giardiosis is a worldwide intestinal parasitosis, affecting both humans and animals. Treatment in dogs remains limited and the lack of efficacy of the few approved medications is a rising concern. In this study, 23 dogs raised by veterinary students and naturally infected with Giardia duodenalis were treated in home conditions with fenbendazole (50 mg/kg orally for 5 consecutive days). Fecal samples were collected immediately before treatment (FS1), 2-4 days after treatment (FS2) and 8-10 days after treatment (FS3). Giardia duodenalis cyst excretion was measured quantitatively by direct immunofluorescence assay (DFA) at FS1, FS2 and FS3. Molecular typing with a nested PCR targeting the SSU rDNA locus was also performed at FS1 and FS2. Fecal consistency improved in 16/21 dogs (76%) and mean cyst shedding was reduced by 84% after treatment. However, only 8/23 dogs (35%) achieved therapeutic success (≥90% reduction of cysts) and only 4/23 dogs (17%) had complete elimination of G. duodenalis. Molecular typing showed that dogs harbored only canine-specific assemblages, with a high prevalence of assemblage C in analyzed samples (30/39). We also detected different assemblages after treatment and nucleotide substitutions in assemblage C sequences that have not been described previously. Eight to ten days after treatment, high Giardia cyst excretion was measured, suggesting possible reinfection despite hygiene measures and/or multiplication. These data suggest that fenbendazole treatment may improve fecal consistency but has limited therapeutic efficacy against giardiosis in this population of dogs. Further research is still needed to assess the efficacy of fenbendazole against canine giardiosis.

Title: Absence d'efficacité du fenbendazole contre Giardia duodenalis dans une population de chiens naturellement infectés en France. Abstract: La giardiose est une parasitose intestinale mondiale, touchant à la fois l'homme et les animaux. Chez le chien, le traitement reste limité et le manque d'efficacité des quelques médicaments autorisés inquiète de plus en plus. Dans cette étude, 23 chiens d'étudiants vétérinaires et infectés naturellement par Giardia duodenalis ont été traités en conditions réelles avec du fenbendazole (50 mg/kg par voie orale pendant 5 jours consécutifs). Des échantillons de selles ont été collectés juste avant le traitement (FS1), 2­4 jours après traitement (FS2) et 8­10 jours après traitement (FS3). L'excrétion de kystes de G. duodenalis a été mesurée quantitativement par immunofluorescence directe (IFD) à FS1, FS2 et FS3. Un génotypage par PCR nichée ciblant le locus SSU ADNr a également été réalisé à FS1 et FS2. La consistance des selles a été améliorée chez 16/21 (76 %) chiens et la moyenne d'excrétion des kystes a été réduite de 84 % juste après le traitement. Seulement 8/23 (35 %) chiens ont atteint un succès thérapeutique (≥ 90 % de réduction d'excrétion de kystes) et 4/23 (17 %) chiens ont eu une élimination complète de G. duodenalis. L'analyse des séquences a montré que les chiens présentaient seulement des assemblages génotypiques spécifiques de l'espèce canine, avec une forte prévalence de l'assemblage C dans les échantillons analysés (30/39). Des changements d'assemblage après traitement et des substitutions nucléotidiques jamais décrites au sein de l'assemblage C ont également été observés. Huit à dix jours après traitement, une forte excrétion de kystes de G. duodenalis a été mesurée : malgré les mesures hygiéniques, une réinfection et/ou une multiplication semblent probables. Ces données suggèrent que le traitement au fenbendazole peut améliorer la consistance des selles mais a une efficacité thérapeutique limitée contre la giardiose dans cette population de chiens. Des recherches supplémentaires sont encore nécessaires pour évaluer l'efficacité du fenbendazole contre la giardiose canine.