Giardia-infected Mice Research Articles

Giardia spp.-induced microbiota dysbiosis disrupts intestinal mucin glycosylation

ABSTRACT Infection with the protozoan parasite Giardia duodenalis (syn. intestinalis, lamblia) has been associated with intestinal mucus disruptions and microbiota dysbiosis. The mechanisms remain incompletely understood. Mucus consists primarily of densely glycosylated mucin glycoproteins. Mucin O-glycans influence mucus barrier properties and mucin–microbe interactions and are frequently altered during disease. In this study, we observed time-dependent and regiospecific alterations to intestinal mucin glycosylation patterns and the expression of mucin-associated glycosyltransferase genes during Giardia infection. Glycosylation alterations were observed in Giardia-infected mice in the upper small intestine, the site of parasite colonization, and in the distal colon, where active trophozoites were absent. Alterations occurred as early as day 2 post-infection and persisted in mice after parasite clearance. We also observed small intestinal goblet cell hyperplasia and thinning of the distal colon mucus barrier during early infection, and microbiota alterations and altered production of cecal SCFAs. Giardia-induced alterations to mucin glycosylation were at least in part dependent on microbiota dysbiosis, as transplantation of a dysbiotic mucosal microbiota collected from Giardia-infected mice recapitulated some alterations. This study describes a novel mechanism by which Giardia alters intestinal mucin glycosylation, and implicates the small intestinal microbiota in regulation of mucin glycosylation patterns throughout the gastrointestinal tract.

Giardial lipid rafts share virulence factors with secreted vesicles and participate in parasitic infection in mice.

Giardia lamblia, a protozoan parasite, is a major cause of waterborne infection, worldwide. While the trophozoite form of this parasite induces pathological symptoms in the gut, the cyst form transmits the infection. Since Giardia is a noninvasive parasite, the actual mechanism by which it causes disease remains elusive. We have previously reported that Giardia assembles cholesterol and GM1 glycosphingolipid-enriched lipid rafts (LRs) that participate in encystation and cyst production. To further delineate the role of LRs in pathogenesis, we isolated LRs from Giardia and subjected them to proteomic analysis. Various cellular proteins including potential virulence factors—e.g., giardins, variant surface proteins, arginine deaminases, elongation factors, ornithine carbomyltransferases, and high cysteine-rich membrane proteins—were found to be present in LRs. Since Giardia secretes virulence factors encapsulated in extracellular vesicles (EVs) that induce proinflammatory responses in hosts, EVs released by the parasite were isolated and subjected to nanoparticle tracking and proteomic analysis. Two types of EV—i.e., small vesicles (SVs; <100 nm, exosome-like particles) and large vesicles (LVs; 100–400 nm, microvesicle-like particles)—were identified and found to contain a diverse group of proteins including above potential virulence factors. Although pretreatment of the parasite with two giardial lipid raft (gLR) disruptors, nystatin (27 μM) and oseltamivir (20 μM), altered the expression profiles of virulence factors in LVs and SVs, the effects were more robust in the case of SVs. To examine the potential role of rafts and vesicles in pathogenicity, Giardia-infected mice were treated with oseltamivir (1.5 and 3.0 mg/kg), and the shedding of cysts were monitored. We observed that this drug significantly reduced the parasite load in mice. Taken together, our results suggest that virulence factors partitioning in gLRs, released into the extracellular milieu via SVs and LVs, participate in spread of giardiasis and could be targeted for future drug development.

A56 TUFT CELL RESPONSES DURING ACUTE- AND LATE-STAGE GIARDIA INFECTION

BackgroundEpithelial tuft cells can detect and respond to enteric infections and appear to help clear some intestinal parasites. Through tuft cell luminal surface receptors, tuft cells can sense ligands directly supplied by a parasite, or indirectly via excretory/secretory products. We hypothesize that microbiome alterations may also modulate tuft cell-derived gut responses. Tuft cells release the alarmin cytokine IL-25 which, upon acting on type 2 innate lymphoid cells (ILC2), ultimately lead to tuft and goblet cell hyperplasia. Upon helminth infections, tuft and goblet cell hyperplasia occurred concurrently, and coincided with the peak of infection. The role of tuft cells in infections with the enteric protozoan parasite Giardia sp. is unknown. The aim of our study is to characterize how tuft cells may be implicated in the pathophysiology of giardiasis, in an attempt to uncover novel regulatory pathways of intestinal physiology.AimsIn this study, we aim to characterise the tuft cell response to Giardia infection during acute and late stages of infection and to assess goblet cell hyperplasia.Methods5–7-week-old C57BL/6 mice and tuft cell-deficient mice ( Pou2f3-/-) were orally gavaged with 5x104Giardia muris trophozoites and scarified at days 4, 11 and 21 post-infection. Parasite burden was assessed in the duodenum. Immunofluorescence (IF) staining of doublecortin-like kinase 1 (DCLK1) – a marker of tuft cells – was performed on C57BL/6 mice jejunum tissue sections and the number of tuft cells was quantified. Goblet cells were quantified in PAS/AB-stained jejunum sections. Quantitative PCR (qPCR) was performed on Dclk1, the epithelial secretory cell transcription factor Atoh1, and the mucus gene Muc2.Results G. muris infected C57BL/6 mice displayed high parasite load at days 4 ( p<0.05) and 11 ( p<0.05), with no or low parasite burden at day 21 ( p<0.05). Pou2f3-/- mice showed less robust parasite burden at days 4 and 11, and similar low parasite burden at day 21, compared to WT mice. At day 21, tuft cell (DCLK1+) counts ( p<0.05) and Dclk1 mRNA expression levels were increased in Giardia infected mice in the jejunum. Goblet cell number and Atoh1 and Muc2 expression were increased at day 4 post-infection.ConclusionsThe data demonstrate that tuft cells expand late in Giardia infection, suggesting that upon parasite infection, tuft cells may possess roles in tissue repair or clearance of infection. Tuft cell-deficient mice ( Pou2f3-/-) had lower parasite burden early in Giardia infection, counter-intuitively suggesting that tuft cells may facilitate trophozoite colonization, further highlighting the novelty of these findings. The crosstalk between tuft cells, Giardia, and other host responses during acute and late stages of infection remain to be fully characterised.Funding AgenciesNSERC

A15 GIARDIA DUODENALIS INDUCES REGIOSPECIFIC ALTERATIONS TO INTESTINAL MUCOSAL GLYCOSYLATION PATTERNS

BackgroundDisruption of intestinal barrier function is important in the pathogenesis of numerous intestinal diseases and can lead to increased intestinal permeability. During infection with the protozoan parasite Giardia duodenalis, intestinal permeability is increased in part due to disruption of the intestinal mucus barrier. Intestinal mucus is composed primarily of the heavily glycosylated MUC2 mucin. Glycosylation of mucins is important for maintaining the structure of the mucus gel, and glycans are key mediators of host-microbe interactions, as they provide binding sites to microbes and can be degraded as a nutrient source. Changes to mucin glycosylation patterns have been noted during intestinal inflammation and bacterial infection, and may contribute to altered intestinal permeability and dysbiosis. We hypothesized that intestinal mucosal glycosylation patterns may be disrupted during Giardia infection, and that this may contribute to Giardia-induced barrier dysfunction.AimsCharacterize changes to mucosal glycosylation patterns in the small and large intestines during in vivo Giardia infection.Methods3–4 week old C57BL/6 mice were infected with Giardia duodenalis strain GS/M for 7 days. Tissue sections from the jejunum and colon were collected and stained with various fluorescein-coupled lectins (CONA, DBA, PNA, WGA, SNA, UEA-1) and fluorescence was quantified and normalized to tissue area. Quantitative PCR (qPCR) was performed for glycosyltransferase genes in the jejunum and colon.ResultsIn the jejunum, abundance of N-acetylglucosamine increased upon infection, while sialic acid and fucose abundance were significantly decreased in infected mice compared to controls. Conversely, in the distal colon, mannose and sialic acid abundance increased significantly upon infection.Expression of mucin-associated glycosyltransferase genes was also altered in the small and large intestines of Giardia infected mice. In the jejunums of infected mice, expression of the sulfotransferase Chst4 decreased, while the fucosyltransferase Fut2 and the sialyltransferase St6GalNAc1 increased in comparison to controls. In both the jejunum and distal colon, expression of the core 2 synthase C2GnT1 increased, while expression of the core 1 synthase C1GalT1 was similar between control and infected mice.ConclusionsGlycosylation patterns and the expression of glycosyltransferase genes are altered in both the small and large intestines of Giardia-infected mice. Disruptions to mucin glycans appear to be regiospecific, suggesting that unique mechanisms are involved in the regulation of mucosal glycosylation during parasitic infection throughout different regions of the gut. These findings uncover a novel mechanism in the pathogenesis of Giardia infection, and may be important in understanding intestinal barrier dysfunction in a variety of different gastrointestinal diseases.Funding AgenciesCCC

Structural and Chemical Alterations to the Intestinal Mucus Barrier during Giardia duodenalis Infection

Background During the course of infection with the protozoan parasite Giardia duodenalis, both mucus production and structure of the mucus layers have been shown to be altered throughout the large and small intestines. This potentially has significant impacts on mucosal barrier function, predisposing a host to mucosal inflammation, which contributes both to Giardia pathogenesis and to development of post-infectious complications. We have attempted to characterize the mechanisms by which Giardia modulates mucus production and secretion by intestinal goblet cells, as well as chemical alterations to mucin proteins that occur in response to Giardia infection. Several effects have been found to be dependent on Giardia cysteine protease activity, suggesting a potential mechanism involving protease activated receptors. Methods The human colonic epithelial cell line LS174T was infected with Giardia trophozoites (isolates NF, WB, S2, and GSM). Prior to infection, trophozoites were treated with E64, a broad-spectrum cysteine protease inhibitor, and LS174T were treated with a pepducin PAR2 antagonist, BAPTA, a calcium chelator, or U0126, an ERK1/2 inhibitor. MUC2 mucin gene expression was assessed using quantitative PCR (qPCR). Wild type (WT) and PAR2 deficient (PAR2 −/−) mice were infected with Giardia trophozoites. The colonic mucus layer was stained using WGA and qPCR was performed for Muc2 and Muc5ac in the small and large intestines. Expression of glycosyltransferase enzymes was assessed using qPCR. Results Giardia NF, S2, and WB, but not GSM, upregulate MUC2 expression in LS174T cells. This increase was attenuated by inhibition of Giardia cysteine proteases and by antagonism of PAR2 or inhibition of calcium release or ERK1/2 signaling in LS174T cells. Giardia infected WT mice show increased Muc2 and Muc5ac expression in the colon, and increased Muc5ac but decreased Muc2 expression in the jejunum. These changes were not seen in PAR2−/− mice. Both WT infected and PAR2−/− non-infected mice showed thinning of the colonic mucus layer compared to WT controls. There was some recovery in thickness in PAR2−/− infected mice. Expression of the fucosyltransferase Fut2 was increased in the jejunums of WT mice, while the sulfotransferase Chst4 was decreased. Expression of the glycan core synthase C2GnT1 was increased in the WT jejunum, while C1GalT1 expression was slightly decreased in the jejunum and colon. Conclusions Giardia-induced alterations to mucin gene expression in vitro and in vivo are dependent on PAR2 signaling. Canonical PAR2 signaling, characterized by calcium release and ERK1/2 pathway activation, contribute to altered expression of the MUC2 mucin in colonic goblet cell cultures. Expression of several glycosyltransferases, responsible for the addition of glycan groups to intestinal mucins, are altered during Giardia infection in vivo, suggesting mucosal alterations may involve chemical modifications of mucin proteins.

Disturbed gut microbiota and bile homeostasis in Giardia-infected mice contributes to metabolic dysregulation and growth impairment.

Although infection with the human enteropathogen Giardia lamblia causes self-limited diarrhea in adults, infant populations in endemic areas experience persistent pathogen carriage in the absence of diarrhea. The persistence of this protozoan parasite in infants has been associated with reduced weight gain and linear growth (height-for-age). The mechanisms that support persistent infection and determine the different disease outcomes in the infant host are incompletely understood. Using a neonatal mouse model of persistent G. lamblia infection, we demonstrate that G. lamblia induced bile secretion and used the bile constituent phosphatidylcholine as a substrate for parasite growth. In addition, we show that G. lamblia infection altered the enteric microbiota composition, leading to enhanced bile acid deconjugation and increased expression of fibroblast growth factor 15. This resulted in elevated energy expenditure and dysregulated lipid metabolism with reduced adipose tissue, body weight gain, and growth in the infected mice. Our results indicate that this enteropathogen's modulation of bile acid metabolism and lipid metabolism in the neonatal mouse host led to an altered body composition, suggesting how G. lamblia infection could contribute to growth restriction in infants in endemic areas.

Western Diet Increases the Severity of Giardia Infection in Association with Gut Microbiota Dysbiosis

AIMSGiardia duodenalis is a protozoan parasite responsible for giardiasis, a leading cause of diarrheal disease worldwide. Giardia sp. infection disrupts the host’s intestinal barrier, leading to acute, chronic, and post‐infectious gut disorders. Giardia infections are prevalent in countries with poor sanitation as well in developed countries, although disease manifestations may vary. Western diet (WD), characterized by a high intake of fat, is known to promote the development of gut inflammatory conditions. In this context, we investigated the impact of short‐term consumption of a WD on the severity and duration of giardiasis. We hypothesized that WD may contribute to the persistence of Giardia in the gut, in association with increased disease severity and microbiota dysbiosis.METHODSC57/BL6 mice were fed for two weeks with 5TJN Western high fat diet (WD) or 5TJS low‐fat (LF) diet prior to infection with Giardia duodenalis GS/M (human isolate). Gut motility and stool water content were assessed before sacrifice at day 7 post infection (PI). Small intestine and colonic sections were collected at day 7 PI for parasite burden and histopathological study. Hypoxia in the colon was assessed by injecting pimonidazole prior to euthanasia. Gut microbiota composition was determined by Illumina Mi‐Seq 16S RNA sequencing. The effect of oleic and palmitic acids (characteristic of HF diets) on Giardia trophozoite metabolic activity was determined by resazurin assay (0.1–0.25 mM). Tight junction disruption in the small intestinal cell line SCNB (dog) infected with G. duodenalis Assemblage A (NF strain) and Assemblage B (GS/M strain) in presence of oleic or palmitic acid (0.3 mM) or both was determined by immunofluorescence.RESULTSGiardia infected C57/BL6 mice fed with WD exhibited a higher trophozoite burden 7 days PI compared to LF infected mice (p&lt;0.001). Giardia infected mice also exhibited enhanced mucosal damage (villus‐crypt ratio) when fed with WD compared to LF infected mice (p&lt;0.01). Increased gut motility (p&lt;0.01) and increased water stool content (p&lt;0.05) were observed in WD infected mice. Giardia infected WD mice exhibited microbiota dysbiosis, with fecal microbiota analysis indicating an increase of Verrucomicrobia and decrease of Firmicutes. Giardia infection caused colonic mucosal normoxia only in mice given a WD. Giardia trophozoite metabolic activity was enhanced in the presence of oleic acid and palmitic acid. Oleic and palmitic acids worsened the disruption of tight junctional proteins (ZO‐1, Claudin‐1 and 4) in non‐transformed intestinal epithelial SCBN cell monolayers infected with G. duodenalis (NF and GS/M isolates).CONCLUSIONThis study gives new insight into how diet may influence the outcome of parasitic diseases. The findings suggest that short term consumption of WD may increase the severity of giardiasis by, at least in part, promoting the growth of Giardia through fatty acid supplementation, predisposing to loss of epithelial integrity, and facilitating microbiota modulation. Therefore, diet intervention such as low‐dietary fat may be beneficial during parasitic enteritis.Support or Funding InformationNSERC

Encapsulation of Low Metronidazole Dose in Poly (D,L-lactide-co-glycolide) (PLGA) Nanoparticles Improves Giardia intestinalis Treatment

BackgroundThe present study was designed to investigate the antigiardial efficacy of low metronidazole dose loaded-D.L-lactide-co-glycolide (LMD-PLGA) nanoparticles (NPs) and to compare it with the standard high dose of metronidazole either free (HMD) or loaded on PLGA (HMD-PLGA).Materials and MethodsPLGA NPs were prepared by single emulsification method, metronidazole (MTZ) was loaded in low and high doses. The nanoparticles were evaluated in vivo for mice model. The Giardia intestinalis infected mice were treated by LMD and HMD either free or PLGA NPs loaded, the parasitic load and ployclonal antigiardial serum antibodies (IgG and IgA) were recorded. Histopathological studies on intestinal and liver sections were applied.ResultsMTZ-PLGA NPs was successfully prepared with 81.68% encapsulation efficiency and with an average particle size of approximately 228.00 ± 43.19 nm and -32.28 ± 0.07 mV Zeta potential. Experimentally, it was observed that Giardia intestinalis infected animals administered with LMD-PLGA had completely eliminated cyst shedding and trophozoite count compared with Giardia-infected mice. Further, it was found that animals belonging to LMD-PLGA group had significantly reduced levels of antigiardial IgA (0.99 ± 0.05) antibodies in serum compared with Giardia-infected. Histopathologyically, also animals belonging to LMD-PLGA treated group had intact mucosal epithelium lining, and normal villi with no detection of G. intestinalis trophozoites. In addition to the less toxic effect on the liver tissue compared to free HMD, HMD-PLGA and infected-untreated groups using Ishak grading system.ConclusionOur study showed that PLGA nanoparticles could be atrial delivery systems for antigiardial drugs to improve their therapeutic efficacy and minimize their side effects that results from frequent dosing.

Effects of Western Diet on Giardiasis: A Role for Fatty Acids and Gut Microbiota in the Persistence and Severity of Giardia Infections

AIMSGiardia duodenalis is a protozoan parasite responsible for giardiasis, one of the most common cause of waterborne diseases. Giardia. sp causes epithelial disruptions as well as microbiota alterations. Giardia infections are common in countries with poor sanitation, where the diet is mostly agrarian and high in fiber content. Intriguingly, in such environments, giardiasis has commonly been reported to cause little or no illness. Giardiasis also occurs in developed countries with western/high fat diets, where infections are a common cause of diarrhea. This sutdy investigated the role of diet on the severity and the duration of giardiasis. We hypothesized that western diet may contribute to the persistence of Giardia in the gut. Experiments assessed the role of fatty acids and gut microbiota in pathophysiology.METHODSC57/BL6 mice were fed for two weeks with 5TJN Western diet or 5TJS Low‐Fat (LF) diet prior to infection with Giardia duodenalis GS/M strain (human isolate). The small intestines were collected at day 7 post infection (PI) for histological study and parasite burden. Gut microbiota composition was determined by Illumina Mi‐Seq 16S RNA sequencing. The effect of Oleic and Palmitic acid on Giardia metabolic activity was determined by resazurin assay. Finally, tight junction disruption of small intestinal epithelial cell line SCNB infected with G. duodenalis Assemblage A (NF strain) and Assemblage B (GS/M strain) in the presence of Oleic, Palmitic acid (0.3 mM) or both was determined by immunofluorescence.RESULTSWe observed that Giardia infected C57/BL6 mice that were fed with 5TJN Western diet exhibited a significantly higher trophozoite burden 7 days PI. Giardia infected mice also exhibited more severe mucosal damage (lower villus‐crypt ratio) when fed with Western diet. Fecal microbiota analysis showed that mice fed with Western Diet exhibit a higher abundance of Verrucomicrobia and a lower abundance of Firmicutes than mice fed LF diet. Upon Giardia infection, a decrease of Bacteroidetes and an increase of Firmicutes was observed. Overall, Giardia infection induced an increase of the alpha diversity in Western mice compared to mice given LF diet. Trophozoite metabolic activity was enhanced in the presence of Oleic acid (0.1 mM) and Palmitic acid (0.25mM). In addition, small intestine cell lines infected with G. duodenalis strain NF and GS/M showed a disruption and/or a rearrangement of tight junction proteins such as ZO‐1, Claudin‐1 and 4 when incubated with Oleic and Palmitic acids (0.3 mM).CONCLUSIONThis study gives insight into how diet may influence the outcome of parasitic diseases and intestinal pathophysiology. The findings suggest that a Western Diet may increase the severity of giardiasis by, at least in part, promoting the growth of Giardia through fatty acid supplementation and microbiota modulation. The potential of diet intervention in treatment against parasitic diseases warrants further research.Support or Funding InformationGrant support: NSERCThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Giardia‐Induced Alterations to Intestinal Mucus Production Involve Protease‐Activated Receptor‐2‐Mediated Activation of MAPK and Calcium Release

IntroductionDisruption of the intestinal mucus layers is associated with increased rates of infection and inflammation and has been implicated in a wide variety of intestinal disorders. Giardia duodenalis has been shown to disrupt mucus production and secretion [1] and may therefore be used as a model system to study regulation of these processes in health and disease.AimsDetermine the mechanisms involved in Giardia mediated disruptions to mucus production by intestinal goblet cells. The roles of protease‐activated receptor‐2 (PAR2) and Giardia cysteine proteases were investigated.MethodsThe human colonic epithelial cell line LS174T was infected with Giardia trophozoites (isolates NF, WB, S2, and GSM). Prior to infection, trophozoites were treated with E64, a broad‐spectrum cysteine protease inhibitor, and LS174T were treated with a pepducin PAR2 antagonist, BAPTA, a calcium chelator, or U0126, an ERK1/2 inhibitor. MUC2 mucin gene expression was assessed using quantitative PCR (qPCR).Chinese hamster ovary cells transfected with nano‐luciferase tagged PAR2 were incubated with Giardia trophozoites. Release of enzymes due to cleavage at the receptor N‐terminus by Giardia resulted in a luminescent signal proportional to the number of cleaved receptors.Wild type (WT) and PAR2 deficient (PAR2 −/−) mice were infected with Giardia trophozoites. The colonic mucus layer was stained using fluorescein‐coupled wheat germ agglutinin, and qPCR was performed for Muc2 and Muc5ac.ResultsGiardia isolates NF, S2, and WB, but not GSM, increased MUC2 expression in LS174T cells. This increase was attenuated by inhibition of Giardia cysteine proteases and by antagonism of PAR2 or inhibition of calcium release or MAPK signaling in LS174T cells.Giardia trophozoites cleaved PAR2 at the N‐terminus, suggesting they are capable of activating the receptor. The amount of cleavage was isolate‐dependent, reflecting differences in protease activity, and cleavage was significantly reduced by E64 treatment for all isolates.Giardia infected WT mice show increased Muc2 and Muc5ac expression in the colon, and increased Muc5ac but decreased Muc2 expression in the jejunum. These changes were not seen in PAR2−/− mice. Both WT infected and PAR2−/− non‐infected mice showed thinning of the colonic mucus layer compared to WT controls. There was some recovery in thickness in PAR2−/− infected mice.ConclusionsResults demonstrate that PAR2 plays a significant role in the regulation of mucin gene expression in mice and a human colonic cell line, and indicate a potential pathway in which Giardia cysteine proteases cleave PAR2 at the N‐terminus, activating canonical PAR2 signaling which involves both calcium release and MAPK activation. These pathways in turn contribute to altered mucin gene expression.Support or Funding InformationCrohn's Colitis Canada (CCC)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Comparative Analysis of Antigiardial Potential of Heat Inactivated and Probiotic Protein of Probiotic Lactobacillus rhamnosus GG in Murine Giardiasis.

The present study was designed to envisage the antigiardial efficacy of killed probiotic and probiotic protein (PP) of Lactobacillus rhamnosus GG in murine giardiasis. Experimentally, it was observed that animal administered either with probiotic protein emulsified with adjuvant (PP(E) + Giardia) or killed probiotic (killed probiotic (i/p) + Giardia) had significantly reduced Giardia cycle with respect to observed severity and duration of giardiasis compared with Giardia-infected mice. Further, it was found that animals belonging to PP(E) + Giardia and killed probiotic (i/p) + Giardia had significantly high levels of antigiardial IgA antibody and nitric oxide both in serum and in intestinal fluid compared with Giardia-infected and counter control mice. Histopathologyically, also animals belonging to PP(E) + Giardia and killed probiotic (i/p) + Giardia animals had intact mucosal epithelium lining, basal crypts, and normal villi along with increased goblet cells compared with severe microvillus atrophy, vacuolated epithelial cells, and ileitis in Giardia-infected mice. This is the first-ever study to demonstrate that prior administration of either killed probiotics or probiotic protein of effective probiotic reduced both the severity and the duration of giardiasis mainly by modulating the gut microbiome and morphology along with mucosal immunity, but animals belonging to PP(E) + Giardia had better response than killed probiotic (i/p) + Giardia suggesting that probiotic components do have adjuvant potential and may be used as the vaccine candidate for gastrointestinal diseases.

Cross-modulation of pathogen-specific pathways enhances malnutrition during enteric co-infection with Giardia lamblia and enteroaggregative Escherichia coli.

Diverse enteropathogen exposures associate with childhood malnutrition. To elucidate mechanistic pathways whereby enteric microbes interact during malnutrition, we used protein deficiency in mice to develop a new model of co-enteropathogen enteropathy. Focusing on common enteropathogens in malnourished children, Giardia lamblia and enteroaggregative Escherichia coli (EAEC), we provide new insights into intersecting pathogen-specific mechanisms that enhance malnutrition. We show for the first time that during protein malnutrition, the intestinal microbiota permits persistent Giardia colonization and simultaneously contributes to growth impairment. Despite signals of intestinal injury, such as IL1α, Giardia-infected mice lack pro-inflammatory intestinal responses, similar to endemic pediatric Giardia infections. Rather, Giardia perturbs microbial host co-metabolites of proteolysis during growth impairment, whereas host nicotinamide utilization adaptations that correspond with growth recovery increase. EAEC promotes intestinal inflammation and markers of myeloid cell activation. During co-infection, intestinal inflammatory signaling and cellular recruitment responses to EAEC are preserved together with a Giardia-mediated diminishment in myeloid cell activation. Conversely, EAEC extinguishes markers of host energy expenditure regulatory responses to Giardia, as host metabolic adaptations appear exhausted. Integrating immunologic and metabolic profiles during co-pathogen infection and malnutrition, we develop a working mechanistic model of how cumulative diet-induced and pathogen-triggered microbial perturbations result in an increasingly wasted host.

Crosstalk between Zinc Status and Giardia Infection: A New Approach.

Zinc supplementation has been shown to reduce the incidence and prevalence of diarrhea; however, its anti-diarrheal effect remains only partially understood. There is now growing evidence that zinc can have pathogen-specific protective effects. Giardiasis is a common yet neglected cause of acute-chronic diarrheal illness worldwide which causes disturbances in zinc metabolism of infected children, representing a risk factor for zinc deficiency. How zinc metabolism is compromised by Giardia is not well understood; zinc status could be altered by intestinal malabsorption, organ redistribution or host-pathogen competition. The potential metal-binding properties of Giardia suggest unusual ways that the parasite may interact with its host. Zinc supplementation was recently found to reduce the rate of diarrhea caused by Giardia in children and to upregulate humoral immune response in Giardia-infected mice; in vitro and in vivo, zinc-salts enhanced the activity of bacitracin in a zinc-dose-dependent way, and this was not due to zinc toxicity. These findings reflect biological effect of zinc that may impact significantly public health in endemic areas of infection. In this paper, we shall explore one direction of this complex interaction, discussing recent information regarding zinc status and its possible contribution to the outcome of the encounter between the host and Giardia.

Dietary zinc supplementation during Giardia lamblia infection enhances growth performance, zinc status and immune response in CD1 mice (392.5)

The effect of zinc (Zn) supplementation for treating diarrhea in children may vary according to the etiological agent. Since Giardia lamblia (G. lamblia) is an important cause of parasitic diarrheal disease worldwide it was of interest to evaluate the effect of different levels of dietary Zn intake on growth performance, Zn status and immune response during infection. CD1 mice were fed 1 of 4 experimental diets: low Zn (ZnL), adequate Zn (ZnA), supplemented Zn (ZnS1) and highly supplemented Zn (ZnS2) diet containing 10, 30, 223 and 1383 mg Zn/kg respectively. After a 10‐day feeding period, mice were orally inoculated with G. lamblia trophozoites and followed on the assigned diet during the course of infection. Giardia‐free mice fed ZnL or ZnA diet were able to attain normal growth while Giardia‐infected mice presented significant growth retardation and lower serum Zn levels. ZnS1 and ZnS2 diets avoided this weight loss during infection, enhanced the production of anti‐G. lamblia IgG and improved serum Zn levels despite the ongoing infection. Maximum growth rate and antibody mediated response were attained in mice fed ZnS1 diet with no further increases by feeding higher Zn levels (ZnS2). These findings reflect biological effect of Zn that could be of public health importance in endemic areas of infection. (Partially published, Nutrients 2013, 5(9), 3447‐3460)Grant Funding Source: Supported by CONACyT

Administration of kefir-fermented milk protects mice against Giardia intestinalis infection

Giardiasis, caused by the protozoan Giardia intestinalis, is one of the most common intestinal diseases worldwide and constitutes an important problem for the public health systems of various countries. Kefir is a probiotic drink obtained by fermenting milk with 'kefir grains', which consist mainly of bacteria and yeasts that coexist in a complex symbiotic association. In this work, we studied the ability of kefir to protect mice from G. intestinalis infection, and characterized the host immune response to this probiotic in the context of the intestinal infection. Six- to 8-week-old C75BL/6 mice were separated into four groups: controls, kefir mice (receiving 1 : 100 dilution of kefir in drinking water for 14 days), Giardia mice (infected orally with 4×10(7) trophozoites of G. intestinalis at day 7) and Giardia-kefir mice (kefir-treated G. intestinalis-infected mice), and killed at 2 or 7 days post-infection. Kefir administration was able to significantly reduce the intensity of Giardia infection at 7 days post-infection. An increase in the percentage of CD4(+) T cells at 2 days post-infection was observed in the Peyer's patches (PP) of mice belonging to the Giardia group compared with the control and kefir groups, while the percentage of CD4(+) T cells in PP in the Giardia-kefir group was similar to that of controls. At 2 days post-infection, a reduction in the percentage of B220-positive major histocompatibility complex class II medium cells in PP was observed in infected mice compared with the other groups. At 7 days post-infection, Giardia-infected mice showed a reduction in RcFcε-positive cells compared with the control group, suggesting a downregulation of the inflammatory response. However, the percentages of RcFcε-positive cells did not differ from controls in the kefir and Giardia-kefir groups. An increase in IgA-positive cells was observed in the lamina propria of the kefir group compared with controls at 2 days post-infection. Interestingly, the diminished number of IgA-positive cells registered in the Giardia group at 7 days post-infection was restored by kefir feeding, although the increase in IgA-positive cells was no longer observed in the kefir group at that time. No significant differences in CXCL10 expression were registered between groups, in concordance with the absence of inflammation in small-intestinal tissue. Interestingly, a slight reduction in CCL20 expression was observed in the Giardia group, suggesting that G. intestinalis might downregulate its expression as a way of evading the inflammatory immune response. On the other hand, a trend towards an increase in TNF-α expression was observed in the kefir group, while the Giardia-kefir group showed a significant increase in TNF-α expression. Moreover, kefir-receiving mice (kefir and Giardia-kefir groups) showed an increase in the expression of IFN-γ, the most relevant Th1 cytokine, at 2 days post-infection. Our results demonstrate that feeding mice with kefir reduces G. intestinalis infection and promotes the activation of different mechanisms of humoral and cellular immunity that are downregulated by parasitic infection, thus contributing to protection.

Oral Administration of the Probiotic Lactobacillus casei Ameliorates Gut Morphology and Physiology in Malnourished-Giardia intestinalis-Infected BALB/c Mice

Malnutrition reduces the host immunity and enhances the host susceptibility to various diseases. The present study describes the effect of oral administration of probiotic Lactobacillus casei to malnourished-Giardia-infected BALB/c mice with respect to surface alterations and brush border membrane enzyme activity of the small intestine. It was observed that probiotic feeding either prior to or simultaneously with Giardia infection to malnourished mice led to significantly enhanced activity of disaccharidases compared with malnourished and Giardia-infected mice. Scanning electron microscopy also revealed less mucosal damage in the villi of small intestine of probiotic-fed malnourished-Giardia-infected mice compared with completely damaged, mummified, or blunted villi of malnourished-Giardia-infected mice. The findings indicate that probiotics can be used as the prophylactic candidate in abrogating the gut and intestinal dissacharidases anamolies in malnourished hosts suffering from the intestinal diseases.

Effects of Dietary Zinc Manipulation on Growth Performance, Zinc Status and Immune Response during Giardia lamblia Infection: A Study in CD-1 Mice

Associations between Giardia lamblia infection and low serum concentrations of zinc have been reported in young children. Interestingly, relatively few studies have examined the effects of different dietary zinc levels on the parasite-infected host. The aims of this study were to compare the growth performance and zinc status in response to varying levels of dietary zinc and to measure the antibody-mediated response of mice during G. lamblia infection. Male CD-1 mice were fed using 1 of 4 experimental diets: adequate-zinc (ZnA), low-zinc (ZnL), high-zinc (ZnH) and supplemented-zinc (ZnS) diet containing 30, 10, 223 and 1383 mg Zn/kg respectively. After a 10 days feeding period, mice were inoculated orally with 5 × 106 G. lamblia trophozoites and were maintained on the assigned diet during the course of infection (30 days). Giardia-free mice fed ZnL diets were able to attain normal growth and antibody-mediated response. Giardia-infected mice fed ZnL and ZnA diets presented a significant growth retardation compared to non-infected controls. Zinc supplementation avoided this weight loss during G. lamblia infection and up-regulated the host’s humoral immune response by improving the production of specific antibodies. Clinical outcomes of zinc supplementation during giardiasis included significant weight gain, higher anti-G. lamblia IgG antibodies and improved serum zinc levels despite the ongoing infection. A maximum growth rate and antibody-mediated response were attained in mice fed ZnH diet. No further increases in body weight, zinc status and humoral immune capacity were noted by feeding higher zinc levels (ZnS) than the ZnH diet. These findings probably reflect biological effect of zinc that could be of public health importance in endemic areas of infection.

Persistent gut barrier damage and commensal bacterial influx following eradication of Giardia infection in mice

BackgroundRecent studies of Giardia lamblia outbreaks have indicated that 40–80% of infected patients experience long-lasting functional gastrointestinal disorders after parasitic clearance. Our aim was to assess changes in the intestinal barrier and spatial distribution of commensal bacteria in the post-clearance phase of Giardia infection.MethodsMice were orogastrically inoculated with G. lamblia trophozoites (strain GS/M) or pair-fed with saline and were sacrificed on post-infective (PI) days 7 (colonization phase) and 35 (post-clearance phase). Gut epithelial barrier function was assessed by Western blotting for occludin cleavage and luminal-to-serosal macromolecular permeability. Gut-associated, superficial adherent, and mucosal endocytosed bacteria were measured by agar culturing and were examined by fluorescence in situ hybridization. Intracellular bacteria cultured from isolated mucosal cells were characterized by 16S rDNA sequencing. Neutrophil-specific esterase staining, a myeloperoxidase activity assay, and enzyme-linked immunosorbent assays for cytokine concentrations were used to verify intestinal tissue inflammation.ResultsTight junctional damage was detected in the intestinal mucosa of Giardia-infected mice on PI days 7 and 35. Although intestinal bacterial overgrowth was evident only during parasite colonization (PI day 7), enhanced mucosal adherence and endocytosis of bacteria were observed on PI days 7 and 35. Multiple bacterial strains, including Bacillus, Lactobacillus, Staphylococcus, and Phenylobacterium, penetrated the gut mucosa in the post-infective phase. The mucosal influx of bacteria coincided with increases in neutrophil infiltration and myeloperoxidase activity on PI days 7 and 35. Elevated intestinal IFNγ, TNFα, and IL-1β levels also were detected on PI day 35.ConclusionsGiardia-infected mice showed persistent tight junctional damage and bacterial penetration, accompanied by mucosal inflammation, after parasite clearance. These novel findings suggest that the host’s unresolved immune reactions toward its own microbiota, due to an impaired epithelial barrier, may partly contribute to the development of post-infective gut disorders.

Lactobacillus rhamnosus GG antagonizes Giardia intestinalis induced oxidative stress and intestinal disaccharidases: an experimental study

The present study describes the in vivo modulatory potential of Lactobacillus rhamnosus GG (LGG), an effective probiotic, in Giardia intestinalis-infected BALB/c mice. Experimentally, it was observed that oral administration of lactobacilli prior or simultaneous with Giardia trophozoites to mice, efficiently (p < 0.05) reduced both the severity and duration of giardiasis. More specifically, probiotics fed, Giardia-infected mice, showed a significant increase in the levels of antioxidants [reduced glutathione (GSH) and superoxide dismutase (SOD)] and intestinal disaccharidases [sucrase and lactase] and decreased levels of oxidants in the small intestine, in comparison with Giardia-infected mice. Histopathological findings also revealed almost normal cellular morphology of the small intestine in probiotic-fed Giardia-infected mice compared with fused enterocytes, villous atrophy and increased infiltration of lymphocytes in Giardia-infected mice. The results of the present study has shed new light on the anti-oxidative properties of LGG in Giardia mediated tissue injury, thereby suggesting that the effects of probiotic LGG are biologically plausible and could be used as an alternative microbial interference therapy.

Probiotic Lactobacillus rhamnosus GG Modulates the Mucosal Immune Response in Giardia intestinalis-Infected BALB/c Mice

Gut homeostasis can be altered by the oral administration of health-promoting microorganisms, namely probiotics that are known to reinforce the host immune response. The aim of this study was to elucidate the immunomodulatory effect of orally administered probiotic Lactobacillus rhamnosus GG (LGG) in Giardia-infected mice. BALB/c mice were fed orally with probiotic LGG either 7 days prior to or simultaneously with the challenge dose of Giardia trophozoites. The administration of the probiotic was continued for 25 days, and immunomodulatory potentials in terms of secretory immunoglobulin A (IgA) levels, CD8+ and CD4+ T lymphocytes, and expression of pro-inflammatory [tumor necrosis factor-alpha, interferon-gamma (INF-γ)] and anti-inflammatory cytokines [interleukin (IL)-4, IL-6, IL-10] were studied. Oral feeding of LGG prior to or simultaneously with the test dose of Giardia seems to have modulated both arms (humoral and cellular) of the mucosal immune system since a significant increase in the levels of specific secretory IgA antibody, IgA+ cells, and CD4+ T lymphocytes were observed in contrast with the decreased percentage of cytotoxic CD8+ T lymphocytes. The stimulated mucosal immune response in probiotic fed Giardia-infected mice was further correlated with the enhanced levels of anti-inflammatory cytokines IL-6 and IL-10 and reduced levels of pro-inflammatory cytokine INF-γ. This is the first study to show that oral administration of the effective probiotic LGG to Giardia infected mice could be used as a bacterio-therapy that restores the normal gut microflora and modulates the mucosal immune response.