Age and Giardia intestinalis Infection Impact Canine Gut Microbiota

Giardia intestinalis is a flagellated protozoan responsible for giardiosis (also called giardiasis in humans), the most prevalent and widespread parasitic infection in humans and mammals worldwide. The intestinal microbiota is highly diverse and any alteration in its composition may impact on the health of the host. While studies on the mouse model of giardiosis described the role of the gut microbiota in host susceptibility to infection by the parasite, little is known about the gut microbiota during natural infections in dogs and particularly in puppies. In this study, we monitored naturally G. intestinalis-infected puppies for 3 months and quantified cyst excretion every 2 weeks. All puppies remained subclinically infected during the sampling period as confirmed by fecal examination. In parallel, we performed 16S Illumina sequencing of fecal samples from the different time points to assess the impact of G. intestinalis infection on gut microbiota development of the puppies, as well as gut health markers of immunity such as fecal IgA and calprotectin. Sequencing results revealed that the canine fecal microbiota of Giardia-infected puppies becomes more complex and less diverse with increasing age. In addition, significant differences in the structure of the microbiota were observed between puppies with high and low Giardia cyst excretion. Chronic subclinical G. intestinalis infection appears to be associated with some detrimental structural changes in the gut microbiota. G. intestinalis-associated dysbiosis is characterized by an enrichment of facultative anaerobic, mucus-degrading, pro-inflammatory species and opportunistic pathogens, as well as a reduction of Lactobacillus johnsonii at specific time points. Calprotectin levels increased with age, suggesting the establishment of chronic low-grade inflammation in puppies. Further work is needed to demonstrate whether these alterations in the canine gut microbiota could lead to a dysbiosis-related disease, such as irritable bowel syndrome (IBS) or inflammatory bowel disease (IBD).

Efficacy of Capparis spinosa Linn Leaf and Fruit Extracts on Giardia Lambia Cysts In vitro

Aims: Giardia lamblia (G. lamblia) is one of the most common intestinal parasites worldwide. There are some side effects and the reports of parasite resistance to metronidazole as the first line treatment of giardiasis. Therefore, it is essential to discover an effect and safe drug to treat giardiasis. Methodology: In this study, the anti-parasitic activity of hydroalcoholic extracts of Capparis spinosa Linn (C. spinosa) leaves and fruits with different concentrations (4 to 0.125 mg/ml) was assessed against human isolates of the G. lamblia cysts and incubated at 37℃. After staining Giardia cysts with 0.1% eosin, the lethal percentage and 50% lethal concentration (LC50) of fruit and leave C. spinosa L extracts and metronidazole on G. lamblia cysts were calculated after 3, 6, 24 and 48 hours. Results: Anti-giardia activity of fruit and leaf extracts of C. spinosa was different between concentrations and time points (p<0.005). The lethal effect of both C. spinosa L extracts and metronidazole increased significantly in a concentration - and time–dependent response (P<0.0001). The highest lethal percentage of G. lamblia cysts was observed a concentration of 4 mg / ml of fruit (100%) and leaf (44%) extracts of C. spinosa L after 48 hours, respectively. LC50 values were 0.38 ± 0.02mg/ml for fruit extract, 2.32 ± 0.1 mg/ml for leaf extract and 0.53 ± 0.03 µg/ml for metronidazole after 48hours. Conclusion: C. spinosa can be effective in eliminating Giardia cyst of contaminated environments and water.

Therapeutic Effect of Chitosan Nanoparticles and Metronidazole in Treatment of Experimentally Giardiasis Infected Hamsters

Background: The present study aimed to assess the therapeutic effect of chitosan nanoparticles and metronidazole against Giardia lamblia as well as evaluate the efficacy of loading metronidazole on chitosan nanoparticles. Methods: This study was carried out at medical Parasitology Department, Faculty of Medicine, Zagazig University and Theodor Bilharz Research institute (TBRI) from February 2019 to February 2020 on 45 hamsters. They were divided into 5 groups 9 hamsters each: Group A non-infected hamsters, Group B infected control group, Group C, D and E infected with G. lamblia and treated with Chitosan nanoparticles (CsNPs), metronidazole (MTZ) and metronidazoleloaded chitosan nanoparticles (MTZ-CsNPs) respectively. Results: The highest percentage of reduction in the Giardia cyst and trophozoite counts were in group that received MTZ-CsNPs (94.69%, 94.29%). Lower percentages of reduction were recorded for MTZ treated group (90.15%, 89.52%) and CsNPs treated group (63.64%, 75.24%). Histopathological examination showed marked healing of intestinal mucosa after treatment with MTZCsNPs. Conclusion: CsNPs showed a therapeutic effect against Giardia infection in hamsters. Loading of metronidazole on chitosan nanoparticles enhanced therapeutic effect of both CsNPs as well as metronidazole.

A Novel Multiprotein Bridging Factor 1-Like Protein Induces Cyst Wall Protein Gene Expression and Cyst Differentiation in Giardia lamblia

The capacity to synthesize a protective cyst wall is critical for infectivity of Giardia lamblia. It is of interest to know the mechanism of coordinated synthesis of three cyst wall proteins (CWPs) during encystation, a differentiation process. Multiprotein bridging factor 1 (MBF1) gene family is a group of transcription coactivators that bridge various transcription factors. They are involved in cell growth and differentiation in yeast and animals, or in stress response in fungi and plants. We asked whether Giardia has MBF1-like genes and whether their products influence gene expression. BLAST searches of the Giardia genome database identified one gene encoding a putative MBF1 protein with a helix-turn-helix domain. We found that it can specifically bind to the AT-rich initiator promoters of the encystation-induced cwp1-3 and myb2 genes. MBF1 localized to cell nuclei and cytoplasm with higher expression during encystation. In addition, overexpression of MBF1 induced cwp1-3 and myb2 gene expression and cyst generation. Mutation of the helixes in the helix-turn-helix domain reduced cwp1-3 and myb2 gene expression and cyst generation. Chromatin immunoprecipitation assays confirmed the binding of MBF1 to the promoters with its binding sites in vivo. We also found that MBF1 can interact with E2F1, Pax2, WRKY, and Myb2 transcription factors that coordinately up-regulate the cwp genes during encystation. Using a CRISPR/Cas9 system for targeted disruption of mbf1 gene, we found a downregulation of cwp1-3 and myb2 genes and decrease of cyst generation. Our results suggest that MBF1 is functionally conserved and positively regulates Giardia cyst differentiation.

A smartphone microscope method for simultaneous detection of (oo)cyst of Cryptosporodium and Giardia

Gastrointestinal disorders caused by ingestion of (oo)cysts of Cryptosporodium and Giardia is one of the major health problems in developing countries. We developed a smartphone based microscopic assay method to screen (oo)cysts of Cryptosporodium and Giardia contamination in vegetable and water samples. We used sapphire ball lens as the major imaging element to modify a smartphone as a microscope. Imaging parameters such as field of view and magnification, and image contrast under different staining and illumination conditions were measured. The smartphone microscope method consisting of ball lens of 1 mm diameter, white LED as illumination source and Lugols’s iodine staining provided magnification and contrast capable of distinguishing (oo)cysts of Crypstopsporodium and Giardia in the same sample. The analytical performance of the method was tested by spike recovery experiments. The spiking recovery experiments performed on cabbage, carrot, cucumber, radish, tomatoes, and water resulted 26.8±10.3, 40.1±8.5, 44.4±7.3, 47.6±11.3, 49.2 ±10.9, and 30.2±7.9% recovery for Cryptosporodium, respectively and 10.2±4.0, 14.1±7.3, 24.2±12.1, 23.2±13.7, 17.1±13.9, and 37.6±2.4 % recovery for Giardia, respectively. These recovery results were found to be similar when compared with the commercial brightfield and fluorescence microscopes. We tested the smartphone microscope system for detecting (oo)cysts on 7 types of vegetable (n=196) and river water (n=18) samples. Forty two percent vegetable and thirty-nine percent water samples were found to be contaminated with Cryptosporodium oocyst. Similarly, thirty one percent vegetable and thirty three percent water samples were contaminated with Giardia cyst. This study showed that the developed method can be a cheaper alternative for simultaneous detection of (oo)cysts in vegetable and water samples.

Investigation of Giardia intestinalis Genotypes among the Food Handlers of Qazvin, Iran

Background: We aimed to investigate the genotypes of Giardia intestinalis among the food handlers in Qazvin, Iran. Methods: Overall, 1530 stool specimens were collected from the food handlers who visited Shahid Bolandian Health Center, Qazvin, Iran during 2016. Specimens were evaluated by microscopic and concentration methods. Twenty specimens with appropriate number of giardia cysts were selected followed by DNA extraction. Determination of giardia genotypes was achieved through PCR and sequencing the glutamate dehydrogenase gene. The phylogenetic tree was drawn using the MEGA7 software. Finally, the data were analyzed statistically with a P-value<0.05 was considered as significant. Results: Twenty stool samples (1.3%) were positive for Giardia cyst. All positive specimens were obtained from male participants with abdominal cramp being their most common symptoms. The mean age for infected individuals was 32 yr. Molecular characterization was successfully performed for 17 isolates and two genotypes A (AII, 65%) and B (BIII, 35%) were identified. Conclusion: The most prevalent giardia genotypes among the food handlers in Qazvin were A (AII) and B (BIII) genotypes with A (AII) genotype as the dominant one in the region. Considering the direct association between the food handlers and public health as well as the impact of geographical and host conditions on dispersion and pathogenicity of various genotypes and their zoonotic aspects, further investigations are necessary.

Whole genome sequencing of dog specific assemblages C and D of Giardia duodenalis from single and pooled cysts indicates host associated genes

Giardia duodenalis (Syn. G. intestinalis or G. lamblia) infects over 280 million people each year and numerous animals. G. duodenalis can be subdivided into 8 assemblages with different host specificity. Unculturable assemblages have so far resisted genome sequencing efforts. In this study we isolated single and pooled cysts of assemblage C and D from dog faeces by FACS and sequenced them using multiple displacement amplification and Illumina paired end sequencing. The genomes of assemblages C and D were compared with genomes of assemblages A and B from humans and assemblage E from ruminants and pigs. The genomes obtained from the pooled cysts and from the single cysts were considered complete (>99% marker genes observed) and the allelic sequence heterozygosity (ASH) of assemblage C and D was 0.89% and 0.74%, respectively. Higher than for assemblage B (> 0.43%) and much higher than for assemblages A and E (<0.01%). The flavohemoglobin and 4Fe-4S binding domain family gene involved in O2 and NO detoxification were only present in assemblages A, B and E. Cathepsin-B orthologs were found in all genomes. Six clades of cathepsin-B orthologs contained one gene of each genome, while in three clades not all assemblages were represented. We conclude that whole genome sequencing from a single Giardia cyst results in complete draft genomes making the genomes of unculturable Giardia assemblages accessible. Observed differences between the genomes of assemblage C and D on one hand and the assemblages A, B and E on the other hand are possibly associated with host specificity.

Detection of duodenal giardia using giemsa stain

The objectives of this study are to evaluate the benefit of using Giemsa staining in endoscopic duodenal biopsies if it provides an appropriate tool to diagnose giardia parasite more accurate than the usual hematoxylin-eosin stain. Method: a retrospective study including fifty patients have been nominated with upper abdominal pain, attended gastrointestinal tract(GIT) consultation unit in medical City of Imam Al-Hussein-Karbala/ Iraq complaining from upper abdominal discomfort. They have been examined by esophagio- gastro duodenoscopy. All duodenal biopsies have also been collected from pathology excluding all celiac disease cases. All biopsies have paraffin and stained with hematoxylin and eosin looking for Giardia; in the following, the slides are stained with Giemsa stain following the manufacture-instruction. Results: 50patients (27 females and 23 males) have been examined depending on the hematoxylin and eosin staining, respectively, the diagnosis is non-specific chronic duodenitis with low MARSH score. Eleven patients from total fifty-two ones (21.15%) have shown positive symptoms for giardia cyst and trophozoite using the Giemsa stain. Conclusion: Giardia infection is difficult to be diagnosed with hematoxylin-eosin stain, especially with low infection. Consequently, Giemsa stain might be used to increase the efficacy of histopathological diagnosis of the parasite.