Multiplex PCR for gastrointestinal parasites in stool: Benchmarking against direct microscopy and simplex PCR.

PURPOSE: To develop and validate a multiplex conventional PCR assay to simultaneously detect Cryptosporidium spp., Entamoeba histolytica, and Giardia lamblia in diarrheal samples as a rapid, cost-effective, and sensitive diagnostic tool for prevalent co-infections for improved diagnostic accuracy and efficiency in resource-limited settings. METHODS: Stool samples collected from patients with gastrointestinal symptoms after taking written consent, processed via wet mount, iodine mount, and PCR assays. Cohen's kappa statistical analysis was done to test agreement. RESULT: Among 240 patients, 28.75% showed intestinal protozoa via Microscopy; Single-plex and multiplex PCR demonstrated 100% concordance, detecting 27.9%; confirmed by sequencing. Highest parasite positivity was observed in transplant and immunocompromised patients, with moderate to almost perfect agreement between microscopy and molecular methods. CONCLUSION: Multiplex-conventional PCR offers superior sensitivity and specificity over microscopy and 100% concordance with single-plex PCR, enabling rapid, cost-effective diagnosis of multiple parasites from single stool sample. Its adoption could revolutionize parasitic infection management in routine diagnostics.

Comprehensive analysis of flavohemoprotein copy number variation in Giardia intestinalis: exploring links to metronidazole resistance.

BACKGROUND: Giardiasis, caused by the protozoan parasite Giardia intestinalis, often presents a treatment challenge, particularly in terms of resistance to metronidazole. Despite extensive research, markers for metronidazole resistance have not yet been identified. METHODS: This study analysed 28 clinical samples of G. intestinalis from sub-assemblage AII, characterised by varying responses to metronidazole treatment. We focussed on copy number variation (CNV) of the multi-copy flavohemoprotein gene, analysed using digital polymerase chain reaction (dPCR) and next generation sequencing (NGS). Additionally, chromosomal ploidy was tested in 18 of these samples. Flavohemoprotein CNV was also assessed in 17 samples from other sub-assemblages. RESULTS: Analyses revealed variable CNVs of the flavohemoprotein gene among the isolates, with no correlation to clinical metronidazole resistance. Discrepancies in CNVs detected from NGS data were attributed to biases linked to the whole genome amplification. However, dPCR helped to clarify these discrepancies by providing more consistent CNV data. Significant differences in flavohemoprotein CNVs were observed across different G. intestinalis sub-assemblages. Notably, Giardia exhibits a propensity for aneuploidy, contributing to genomic variability within and between sub-assemblages. CONCLUSIONS: The complexity of the clinical metronidazole resistance in Giardia is influenced by multiple genetic factors, including CNVs and aneuploidy. No significant differences in the CNV of the flavohemoprotein gene between isolates from metronidazole-resistant and metronidazole-sensitive cases of giardiasis were found, underscoring the need for further research to identify reliable genetic markers for resistance. We demonstrate that dPCR and NGS are robust methods for analysing CNVs and provide cross-validating results, highlighting their utility in the genetic analyses of this parasite.

Microbial Health Risks of Cryptosporidium parvum and Giardia lamblia in Tropical Coastal Water in Araromi, Nigeria.

Objective: Giardia and Cryptosporidium are enteric protozoa that can cause a variety of gastrointestinal diseases, especially in vulnerable people like children, the elderly, and those with impaired immune systems. In order to ascertain the microbiological quality of the recreational water from Araromi Beach in Ilaje Local Government Area, Ondo State, Nigeria. This risk assessment is of great significance to human health protection against waterborne diseases. The aim of this study was to determine the microbial quality of recreational water from Araromi Beach in Ilaje Local Government Area, Ondo State, Nigeria. Methods: Microscopic examination of Cryptosporidium and Giardia oocysts were done. Results: Results revealed maximum occurrence of Cryptosporidium parvum (20 oocysts/100 mL) of water sample in the month of April and maximum occurrence of Giardia lamblia (300 cysts/100 mL) of water sample in the month of June. Additionally, according to Kolmogorov-Smirnov tests for normalcy Ho =0.05, Giardia lamblia and Cryptosporidium parvum were not regularly distributed in the water samples collected from the beach throughout the study period. The average likelihood of contracting Giardia lamblia and Cryptosporidium parvum infections after consuming 100 mL of beach water was 0.96 and 0.35, respectively. The risks of infection associated with Cryptosporidium parvum was lower than those associated with Giardia lamblia in water from the beach, but were both above the acceptable risk limit of 10-4. Conclusion: The results of this study indicate that Giardia and Cryptosporidium may represent serious health hazards to people who engage in aquatic activities. Adopting a comprehensive strategy that includes regular inspections, enhanced detection techniques, and the prevention of aquatic environment pollution may provide clean and safe recreational water for all, thereby safeguarding the public's health.

Correlation between Melatonin and Colostral Regulatory T Cells in Giardia lamblia Infection.

Giardiasis is a parasitic disease caused by Giardia lamblia (G. lamblia) that affects people worldwide. Still, few studies report on the immunoregulatory effects of the biomolecules of colostrum during interactions with G. lamblia. This study aimed to assess the concentrations of melatonin and cortisol hormones, the percentage of Treg cells, and the levels of cytokines IL-10 and TGF-ß in colostrum from mothers who tested positive for the parasite. This cross-sectional study analyzed colostrum samples from 25 puerperal. The samples were tested using an ELISA to determine if they were seropositive for G. lamblia and the type of antibody present (IgM and IgG). Based on the results, the samples were divided into three groups: a control group (N = 10) with no reaction to either IgM or IgG, a group seropositive for IgG (IgG+/IgM-; N = 8), and a group seropositive for IgM (IgM+/IgG-; N = 7). The concentrations of melatonin and cortisol were measured using the ELISA method. Additionally, cytokines IL-10 and TGF-ß and immunophenotyping were analyzed using flow cytometry. In the group that tested positive for IgM anti-G. lamblia, the concentration of melatonin was lower. However, in the colostrum from mothers who tested positive for IgG anti-G. lamblia, the level of this hormone had increased. The cortisol levels were similar between the groups, regardless of seropositivity. There was a higher percentage of Treg cells in the colostrum from mothers who tested positive for IgM anti-G. lamblia. TGF-ß levels also increased in the colostrum of mothers who tested positive for IgM anti-G. lamblia. In the seronegative group for G. lamblia, there was a positive correlation between melatonin concentration and the percentage of Treg cells. These data suggest that the increase in regulatory cells and cytokines and the reduction in melatonin in colostrum from mothers with recent giardia infection may contribute to the evolution and manifestation of the disease.

Postnatal zinc deficiency due to giardiasis disrupts hippocampal and cerebellar development.

BACKGROUND: Giardiasis and zinc deficiency have been identified as serious health problems worldwide. Although Zn depletion is known to occur in giardiasis, no work has investigated whether changes occur in brain structures. METHODS: Three groups of gerbils were used: control (1), orogastrically inoculated on day 3 after birth with trophozoites of two isolates of Giardia intestinalis (HGINV/WB) group (2 and 3). Estimates were made at five ages covering: establishment of infection, Giardia population growth, natural parasite clearance and a post-infection age. QuantiChrome zinc assay kit, cresyl violet staining and TUNEL technique were used. RESULTS: A significant decrease (p<0.01) in tissue zinc was observed and persisted after infection. Cytoarchitectural changes were observed in 75% of gerbils in the HGINV or WB groups. Ectopic pyramidal neurons were found in the cornus ammonis (CA1-CA3). At 60 and 90 days of age loss of lamination was clearly visible in CA1. In the dentate gyrus (DG), thinning of the dorsal lamina and abnormal thickening of the ventral lamina were observed from 30 days of age. In the cerebellum, we found an increase (p<0.01) in the thickness of the external granular layer (EGL) at 14 days of age that persisted until day 21 (C 3 ± 0.3 µm; HGINV 37 ± 5 µm; WB 28 ± 3 µm); Purkinje cell population estimation showed a significant decrease; a large number of apoptotic somas were observed scattered in the molecular layer; in 60 and 90 days old gerbils we found granular cell heterotopia and Purkinje cell ectopia. The pattern of apoptosis was different in the cerebellum and hippocampus of parasitized gerbils. CONCLUSION: The morphological changes found suggest that neuronal migration is affected by zinc depletion caused by giardiasis in early postnatal life; for the first time, the link between giardiasis-zinc depletion and damaged brain structures is shown. This damage may explain the psychomotor/cognitive delay associated with giardiasis. These findings are alarming. Alterations in zinc metabolism and signalling are known to be involved in many brain disorders, including autism.

Unveiling the presence and genotypic diversity of Giardia duodenalis on large-scale sheep farms: insights from the Henan and Ningxia Regions, China.

BACKGROUND: The parasitic protozoan Giardia duodenalis is an important cause of diarrheal disease in humans and animals that can be spread by fecal-oral transmission through water and the environment, posing a challenge to public health and animal husbandry. Little is known about its impact on large-scale sheep farms in China. In this study we investigated G. duodenalis infection of sheep and contamination of the environment in large-scale sheep farms in two regions of China, Henan and Ningxia. METHODS: A total of 528 fecal samples, 402 environmental samples and 30 water samples were collected from seven large-scale sheep farms, and 88 fecal samples and 13 environmental samples were collected from 12 backyard farms. The presence of G. duodenalis was detected by targeting the ß-giardin (bg) gene, and the assemblage and multilocus genotype of G. duodenalis were investigated by analyzing three genes: bg, glutamate dehydrogenase (gdh) and triphosphate isomerase (tpi). RESULTS: The overall G. duodenalis detection rate was 7.8%, 1.4% and 23.3% in fecal, environmental and water samples, respectively. On the large-scale sheep farms tested, the infection rate of sheep in Henan (13.8%) was found to be significantly higher than that of sheep in Ningxia (4.2%) (P < 0.05). However, the difference between the rates of environmental pollution in Henan (1.9%) and Ningxia (1.0%) was not significant (P > 0.05). Investigations of sheep at different physiological stages revealed that late pregnancy ewes showed the lowest infection rate (1.7%) and that young lambs exhibited the highest (18.8%). Genetic analysis identified G. duodenalis belonging to two assemblages, A and E, with assemblage E being dominant. A total of 27 multilocus genotypes were identified for members of assemblage E. CONCLUSIONS: The results suggest that G. duodenalis is prevalent on large-scale sheep farms in Henan and Ningxia, China, and that there is a risk of environmental contamination. This study is the first comprehensive examination of the presence of G. duodenalis on large-scale sheep farms in China. Challenges posed by G. duodenalis to sheep farms need to be addressed proactively to ensure public health safety.

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.

Prevalence and molecular characterization of Giardia duodenalis in companion dogs, domestic livestock and wildlife in the Jordan Basin, Israel.

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

Diversity of gastrointestinal parasites and molecular characterization of Giardia duodenalis in free-living and captive howler monkeys (Alouatta guariba clamitans) in southern Brazil.

Non-human primates (NHPs) are the group that most share infectious agents with humans due to their close taxonomic relationship. The southern brown howler monkeys (Alouatta guariba clamitans) are endemic primates from Brazil and Argentina's Atlantic Forest. This study aimed to investigate the presence of intestinal parasites in free-living (FL) and captive (CA) southern brown howler monkeys. Thirty-nine stool samples were collected in two areas in southern Brazil, 15 FL and 24 CA. Stool sediments obtained by centrifugal sedimentation technique were used for microscopic analysis and direct immunofluorescence assay and evaluated by molecular analysis through amplification and sequencing of TPI fragments. Intestinal parasites Giardia duodenalis, Cryptosporidium spp., and Trypanoxyuris minutus were detected at coproparasitological analysis. This is the first report of the presence of Cryptosporidium spp. in free-living howlers. The molecular characterization of G. duodenalis isolates indicated assemblage B for the first time found in free-living A. guariba clamitans. The high prevalence of G. duodenalis transmission in CA howler monkeys can be explained by direct contact with humans and frequent soil contact. The presence of a potentially zoonotic assemblage in these animals indicates that the process of fragmentation and cohabitation with humans and livestock affects the wildlife, thus indicating a need for eco-health measures.

Mucosal vaccination in a murine gnotobiotic model of Giardia lamblia infection.

Giardia lamblia is an important protozoan cause of diarrheal disease worldwide, delayed development and cognitive impairment in children in low- and middle-income countries, and protracted post-infectious syndromes in developed regions. G. lamblia resides in the lumen and at the epithelial surface of the proximal small intestine but is not mucosa invasive. The protozoan parasite is genetically diverse with significant genome differences across strains and assemblages. Animal models, particularly murine models, have been instrumental in defining mechanisms of host defense against G. lamblia, but mice cannot be readily infected with most human pathogenic strains. Antibiotic pretreatment can increase susceptibility, suggesting that the normal microbiota plays a role in controlling G. lamblia infection in mice, but the broader implications on susceptibility to diverse strains are not known. Here, we have used gnotobiotic mice to demonstrate that robust intestinal infection can be achieved for a broad set of human-pathogenic strains of the genetic assemblages A and B. Furthermore, gnotobiotic mice were able to eradicate infection with a similar kinetics to conventional mice after trophozoite challenge. Germ-free mice could also be effectively immunized by the mucosal route with a protective antigen, α1-giardin, in a manner dependent on CD4 T cells. These results indicate that the gnotobiotic mouse model is powerful for investigating acquired host defenses in giardiasis, as the mice are broadly susceptible to diverse G. lamblia strains yet display no apparent defects in mucosal immunity needed for controlling and eradicating this lumen-dwelling pathogen.

Enteric parasites Cyclospora cayetanensis and Cryptosporidium hominis in domestic and wildlife animals in Ghana.

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

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.

Selection of ssDNA aptamers and construction of aptameric electrochemical biosensor for the detection of Giardia intestinalis trophozoite protein.

Giardia intestinalis is one of the most widespread intestinal parasites and is considered a major cause of epidemic or sporadic diarrhea worldwide. In this study, we aimed to develop a rapid aptameric diagnostic technique for G. intestinalis infection. First, the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process generated DNA aptamers specific to a recombinant protein of the parasite's trophozoite. Ten selection rounds were performed; each round, the DNA library was incubated with the target protein conjugated to Sepharose beads. Then, the unbound sequences were removed by washing and the specific sequences were eluted and amplified by Polymerase Chain Reaction (PCR). Two aptamers were selected, and the dissociation constants (Kd), were determined as 2.45 and 16.95 nM, showed their high affinity for the G. intestinalis trophozoite protein. Subsequently, the aptamer sequence T1, which exhibited better affinity, was employed to develop a label-free electrochemical biosensor. A thiolated aptamer was covalently immobilized onto a gold screen-printed electrode (SPGE), and the binding of the targeted protein was monitored using square wave voltammetry (SWV). The developed aptasensor enabled accurate detection of the G. intestinalis recombinant protein within the range of 0.1 pg/mL to 100 ng/mL, with an excellent sensitivity (LOD of 0.35 pg/mL). Moreover, selectivity studies showed a negligible cross-reactivity toward other proteins such as bovine serum albumin, globulin, and G. intestinalis cyst protein.

Harnessing the power of new genetic tools to illuminate Giardia biology and pathogenesis.

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

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.

Promising effects of 1,8 Cineole to control Giardia lamblia infection: Targeting the inflammation, oxidative stress, and infectivity.

Reportedly, synthetic drugs such as metronidazole, furazolidone, tinidazole, and quinacrine are used for the treatment of giardiasis but are associated with adverse effects. In this study, we aimed to investigate the in vitro and in vivo effects of eucalyptol (ECT, 1,8 cineole) alone and in combination with metronidazole (MNZ) on Giardia lamblia. The effects of ECT on cell viability, plasma membrane permeability, and gene expression levels of adenylate cyclase (AK) and extracellular signal kinases 1 and 2 (ERK1 and ERK2) in trophozoites of G. lamblia were assessed. In vivo, the effects of ECT alone and in combination with MNZ were assessed on mice infected with G. lamblia. In addition, the gene expression of inflammatory genes (e.g., TNF-α, IL-1ß, and IL-10) and antioxidant genes (catalase (CAT), superoxide dismutase 1 (SOD1), glutathione peroxidase 2 (GPX2)) was determined by real-time PCR. The IC50 values of ECT, MNZ, and ECT+MNZ on trophozoites were 30.2 µg/mL, 21.6 µg/mL, and 8.5 µg/mL, respectively. The estimated Fractional inhibitory concentration index (FICI) values for ECT and MNZ were 0.28 and 0.39, respectively. The application of ECT on G. lamblia trophozoites resulted in a dose-dependent increase in plasma membrane permeability, particularly at concentrations of ½ IC50 and IC50 (P < 0.05). The treatment of infected mice with various doses of ECT, mainly in combination with MNZ for 7 days, resulted in a significant decrease (P < 0.001) in the average number and viability of cysts. ECT, especially when combined with MNZ, caused a significant (P < 0.001) reduction in the expression of TNF-α and IL-6 genes, and an increase (P < 0.05) in the expression of IL-10 genes. ECT alone and mainly in combination with MNZ leads to a significant (P < 0.001) increase in the gene expression of CAT, SOD, and GPX genes. These findings demonstrate that the use of ECT in these doses, even for 14 days, does not have any toxic effects on the function of vital liver and kidney tissues. The study findings confirmed the promising effects of ECT against G. lamblia infection both in vitro and in vivo. Considering the possible mechanisms, ECT increases plasma membrane permeability and reduces the expression levels of infectivity-related genes. In addition, ECT suppresses inflammation and oxidative stress, controlling giardiasis in mice. More studies are needed to clarify these findings.

Prevalence and molecular characterization of Cryptosporidium spp., Enterocytozoon bieneusi, and Giardia duodenalis in dairy cattle in Ningxia, northwestern China.

Cryptosporidium spp., Enterocytozoon bieneusi, and Giardia duodenalis are common intestinal pathogens that infect humans and animals. To date, research regarding these three protozoa in the Ningxia Hui Autonomous Region (Ningxia) has mostly been limited to a single pathogen, and comprehensive data on mixed infections are unavailable. This study aimed to evaluate the zoonotic potential of these three protozoa. In this study, small subunit ribosomal RNA (SSU rRNA) and 60 kDa glycoprotein (gp60) genes of Cryptosporidium; internal transcribed spacer (ITS) gene of E. bieneusi; and SSU rRNA, glutamate dehydrogenase (gdh), triosephosphate isomerase (tpi), and beta-giardin (bg) genes of G. duodenalis were examined. DNA extraction, polymerase chain reaction, and sequence analysis were performed on fecal samples collected from 320 dairy cattle at three intensive dairy farms in Ningxia in 2021 to determine the prevalence and genetic characteristics of these three protozoa. The findings revealed that 61.56% (197/320) of the samples were infected with at least one protozoan. The overall prevalence of Cryptosporidium was 19.38% (62/320), E. bieneusi was 41.56% (133/320), and G. duodenalis was 29.38% (94/320). This study identified four Cryptosporidium species (C. bovis, C. andersoni, C. ryanae, and C. parvum) and the presence of mixed infections with two or three Cryptosporidium species. C. bovis was the dominant species in this study, while the dominant C. parvum subtypes were IIdA15G1 and IIdA20G1. The genotypes of E. bieneusis were J, BEB4, and I alongside the novel genotypes NX1-NX8, all belonging to group 2, with genotype J being dominant. G. duodenalis assemblages were identified as assemblages E, A, and B, and a mixed infection involving assemblages A + E was identified, with assemblage E being the dominant one. Concurrently, 11 isolates formed 10 different assemblage E multilocus genotypes (MLGs) and 1 assemblage A MLG and assemblage E MLGs formed 5 subgroups. To the best of our knowledge, this is the first report on mixed infection with two or three Cryptosporidium species in cattle in Ningxia and on the presence of the C. parvum subtype IIdA20G1 in this part of China. This study also discovered nine genotypes of E. bieneusis and novel features of G. duodenalis assemblages in Ningxia. This study indicates that dairy cattle in this region may play a significant role in the zoonotic transmission of Cryptosporidium spp., E. bieneusi, and G. duodenalis.

First report of Giardia duodenalis in dairy cattle and beef cattle in Shanxi, China.

BACKGROUND: Giardia duodenalis is an important intestinal parasitic protozoan that infects several vertebrates, including humans. Cattle are considered the major source of giardiasis outbreak in humans. This study aimed to investigate the prevalence and multilocus genotype (MLG) of G. duodenalis in Shanxi, and lay the foundation for the prevention and control of Giardiosis. METHODS AND RESULTS: DNA extraction, nested polymerase chain reaction, sequence analysis, MLG analysis, and statistical analysis were performed using 858 bovine fecal samples from Shanxi based on three gene loci: ß-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi). The overall prevalence of G. duodenalis was 28.3%, while its prevalence in Yingxian and Lingqiu was 28.1% and 28.5%, respectively. The overall prevalence of G. duodenalis in dairy cattle and beef cattle was 28.0% and 28.5%, respectively. G. duodenalis infection was detected in all age groups evaluated in this study. The overall prevalence of G. duodenalis in diarrhea and nondiarrhea samples was 32.4% and 27.5%, respectively, whereas that in intensively farmed and free-range cattle was 35.0% and 19.9%, respectively. We obtained 83, 53, and 59 sequences of bg, gdh, and tpi in G. duodenalis, respectively. Moreover, assemblage A (n = 2) and assemblage E (n = 81) by bg, assemblage A (n = 1) and assemblage E (n = 52) by gdh, and assemblage A (n = 2) and assemblage E (n = 57) by tpi were identified. Multilocus genotyping yielded 29 assemblage E MLGs, which formed 10 subgroups. CONCLUSIONS: To the best of our knowledge, this is the first study to report cattle infected with G. duodenalis in Shanxi, China. Livestock-specific G. duodenalis assemblage E was the dominant assemblage genotype, and zoonotic sub-assemblage AI was also detected in this region.

Minimal zoonotic risk of cryptosporidiosis and giardiasis from frogs and reptiles.

The zoonotic potential of the protist parasites Cryptosporidium spp. and Giardia duodenalis in amphibians and reptiles raises public health concerns due to their growing popularity as pets. This review examines the prevalence and diversity of these parasites in wild and captive amphibians and reptiles to better understand the zoonotic risk. Research on Giardia in both groups is limited, and zoonotic forms of Cryptosporidium or Giardia have not been reported in amphibians. Host-adapted Cryptosporidium species dominate in reptiles, albeit some reptiles have been found to carry zoonotic (C. hominis and C. parvum) and rodent-associated (C. tyzzeri, C. muris and C. andersoni) species, primarily through mechanical carriage. Similarly, the limited reports of Giardia duodenalis (assemblages A, B and E) in reptiles may also be due to mechanical carriage. Thus, the available evidence indicates minimal zoonotic risk associated with these organisms in wild and captive frogs and reptiles. The exact transmission routes for these infections within reptile populations remain poorly understood, particularly regarding the importance of mechanical carriage. Although the risk appears minimal, continued research and surveillance efforts are necessary to gain a more comprehensive understanding of the transmission dynamics and ultimately improve our ability to safeguard human and animal health.