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.

The Influence of the Protozoan Giardia lamblia on the Modulation of the Immune System and Alterations in Host Glucose and Lipid Metabolism.

Giardia lamblia, the cause of giardiasis, significantly impacts patients with metabolic disorders related to insulin resistance (IR). Both giardiasis and metabolic disorders share elements such as chronic inflammation and intestinal dysbiosis, which substantially affect the metabolic and cytokine profiles of patients. This review discusses the mechanisms of virulence of G. lamblia, its influence on the immune system, and its association with metabolic disorders. The review aims to show how G. lamblia invasion acts on the immune system and the glucose and lipid metabolism. Key findings reveal that G. lamblia infection, by disrupting intestinal permeability, alters microbiota composition and immune responses, potentially impairing metabolic status. Future research should focus on elucidating the specific mechanisms by which G. lamblia influences the metabolism, exploring the long-term consequences of chronic infection, and developing targeted therapeutic strategies that include both parasitic and metabolic aspects. These insights underscore the need for a multidisciplinary approach to the treatment of giardiasis in patients with metabolic disorders.

Effect of dexamethasone on experimental enteritis produced by Giardia lamblia in a Meriones unguiculatus model.

Our aim was to evaluate the impact of immunosuppression on the development of giardiasis. Thirty-six gerbils (4-6 weeks old) were distributed in four groups containing nine animals each: Control (CT); Control-Infected by Giardia lamblia (CTIn), Immunosuppressed (IS), and Immunosuppressed-Infected by G. lamblia (ISIn). Animals in the IS and ISIn groups received intramuscular dexamethasone solution for 25 days. On the 11th day, the animals in the CTIn and ISIn groups were inoculated with G. lamblia. After 14 days of infection, the 25th day of the experiment, all groups were euthanized. Four hours after euthanasia, the intestinal permeability was evaluated and sections of the duodenum and spleen were harvested for morphometric and histopathological analyses. Immunosuppressed groups showed a significant increase in intestinal permeability compared to control and infected groups. Considering that the infection can become chronic in immunosuppressed groups, we should be alert to the possibilities of chronic inflammatory changes, both locally and systemically, due to the loss of the intestinal barrier. Lesions were observed in the duodenal mucosa of the gerbils of the CTIn group, with reduced villi size, crypt hyperplasia, edema, and the presence of inflammatory infiltrate in the lamina propria. In the ISIn group, we observed no inflammation, long and intact villi, and a significant increase in the area of intestinal mucins, despite the large number of trophozoites identified. Our results suggest that exacerbation of the immune response has a direct relationship with the appearance of lesions during enteritis produced by G. lamblia in the assessed model.

Differential antibody responses to Giardia lamblia strain variants expressing dissimilar levels of an immunogenic protein.

AIMS: Giardia lamblia is a protozoan parasite that causes giardiasis, one of the most common worldwide gastrointestinal diseases. For rational development of a Giardia vaccine, increasing our understanding of the host-Giardia interaction is crucial. In this study, we analysed the immunogenicity and antigenicity of two G lamblia strain variants [GS and GS-5G8 (+)], which express different levels of the variant-specific surface protein (VSP) 5G8 and also analysed the intestinal histological changes associated with Giardia infection. METHODS AND RESULTS: We evaluated the antibody responses induced by G lamblia strains in infected, reinfected and immunized C3H/HeJ mice using ELISA, flow cytometry, Western blotting and histological analysis. Our results showed that G lamblia GS-5G8 (+) was more immunogenic and antigenic than the GS strain. The antibody response against the GS-5G8 (+) strain primarily recognized 5G8 protein. Serum antibody from infected and reinfected mice exhibited specific agglutination of trophozoites in vitro. GS-5G8 (+)-infected mice showed higher CD19+ infiltrating cell levels compared to GS-infected animals. CONCLUSION: G lamblia strains with different expression levels of an immunogenic antigen (VSP 5G8) induce differential antibody responses. A better understanding of the immunogenic proteins of G lamblia will contribute to the rational development of an effective vaccine against this parasitic disease.

Identification of Conserved Candidate Vaccine Antigens in the Surface Proteome of Giardia lamblia.

Giardia lamblia, one of the most common protozoal infections of the human intestine, is an important worldwide cause of diarrheal disease, malabsorption, malnutrition, delayed cognitive development in children, and protracted postinfectious syndromes. Despite its medical importance, no human vaccine is available against giardiasis. A crude veterinary vaccine has been developed, and experimental vaccines based on expression of multiple variant-specific surface proteins have been reported, but poorly defined vaccine components and excessive antigen variability are problematic for pharmaceutical vaccine production. To expand the repertoire of antigen candidates for vaccines, we reasoned that surface proteins may provide an enriched source of such antigens since key host effectors, such as secretory IgA, can directly bind to such antigens in the intestinal lumen and interfere with epithelial attachment. Here, we have applied a proteomics approach to identify 23 novel surface antigens of G. lamblia that show >90% amino acid sequence identity between the two human-pathogenic genetic assemblages (A and B) of the parasite. Surface localization of a representative subset of these proteins was confirmed by immunostaining. Four selected proteins, uridine phosphorylase-like protein-1, protein 21.1 (GL50803_27925), α1-giardin, and α11-giardin, were subsequently produced in recombinant form and shown to be immunogenic in mice and G. lamblia-infected humans and confer protection against G. lamblia infection upon intranasal immunization in rodent models of giardiasis. These results demonstrate that identification of conserved surface antigens provides a powerful approach for overcoming a key rate-limiting step in the design and construction of an effective vaccine against giardiasis.

Are immunoenzymatic tests for intestinal protozoans reliable when used on archaeological material?

Intestinal protozoans found in ancient human samples have been studied primarily by microscopy and immunodiagnostic assays. However, such methods are not suitable for the detection of zoonotic genotypes. The objectives of the present study were to utilize immunoenzimatic assays for coproantigen detection of Cryptosporidium sp., Giardia duodenalis, and Entamoeba histolytica/Entamoeba dispar in sixty ancient human and animal samples collected from 14 archaeological sites in South America, and to carry out a critical analysis of G. duodenalis according to results obtained from three diagnostic methodologies: microscopy, immunodiagnostic tests (immunoenzymatic and immunofluorescence), and molecular biology (PCR and sequencing). More than half (31/60) of the samples analyzed using immunoenzymatic tests were positive for at least one of the intestinal protozoans, with 46.6% (28/60) corresponding to G. duodenalis, 26.6% (16/60) to Cryptosporidium sp., and 5% (3/60) to E. histolytica/E. dispar. Cryptosporidium sp. and G. duodenalis coinfection was observed in 15% (9/60) of the samples, whereas all three protozoans were found in 5% (3/60) of samples. In the Northeast Region of Brazil, by immunoenzymatic tests there is evidence that G. duodenlais and Cryptosporidium sp. have infected humans and rodents for at least 7150 years. However, for G. duodenalis, the results from the three diagnostic tests were discordant. Specifically, despite the efficiency of the molecular biology assay in the experimental models, G. duodenalis DNA could not be amplified from the ancient samples. These results raise the following question: Are all ancient samples positive for coproantigen of G. duodenalis by immunoenzymatic tests truly positive? This scenario highlights the importance of further studies to evaluate the sensitivity and specificity of the immunoenzymatic method in the archaeological context.

Modulation of human dendritic cell activity by Giardia and helminth antigens.

Giardia duodenalis is a common intestinal protozoan parasite known to modulate host immune responses, including dendritic cell (DC) function. Coinfections of intestinal pathogens are common, and thus, DCs may be concurrently exposed to antigens from multiple parasites. Here, we investigated the effects of G. duodenalis products on human monocyte-derived DC function independently and in combination with helminth antigens (Ascaris suum and Trichuris suis). All antigens individually induced an anti-inflammatory phenotype in DCs, reducing lipopolysaccharide (LPS)-induced interleukin (IL)-6, IL-12p70 and tumour necrosis factor (TNF)-α secretion. G. duodenalis and T. suis products also consistently upregulated IL-10 production. Despite a similar modulation of cytokine secretion, additive effects between Giardia and helminth products were not observed, indicating a dominant effect of a single parasite stimulus and limited interactive effects on DC function. G. duodenalis trophozoites induced rapid apoptosis in DCs, which was not observed with the helminth antigens suggesting that the modulatory effects of G. duodenalis may override that of A. suum and T. suis. Thus, G. duodenalis modulates DC activity by modulating cytokine secretion and/or inducing apoptosis, which may be a parasite-driven mechanism to dampen host immunity and establish chronic infections. The differential mechanisms of DC modulation by intestinal parasites warrant further attention.

Comparison of ELISA, nested PCR and sequencing and a novel qPCR for detection of Giardia isolates from Jordan.

Little is known about the prevalence of Giardia duodenalis in human patients in Jordan and all previous studies have used direct microscopy, which lacks sensitivity. The present study developed a novel quantitative PCR (qPCR) assay at the ß-giardin (bg) locus and evaluated its use as a frontline test for the diagnosis of giardiasis in comparison with a commercially available ELISA using nested PCR and sequencing of the glutamate dehydrogenase (gdh) locus (gdh nPCR) as the gold standard. A total of 96 human faecal samples were collected from 96 patients suffering from diarrhoea from 5 regions of Jordan and were screened using the ELISA and qPCR. The analytical specificity of the bg qPCR assay revealed no cross-reactions with other genera and detected all the Giardia isolates tested. Analytical sensitivity was 1 Giardia cyst per µl of DNA extract. The overall prevalence of Giardia was 64.6%. The clinical sensitivity and specificity of the bg qPCR was 89.9% and 82.9% respectively compared to 76.5 and 68.0% for the ELISA. This study is the first to compare three different methods (ELISA, bg qPCR, nested PCR and sequencing at the gdh locus) to diagnose Jordanian patients suffering from giardiasis and to analyze their demographic data.

Longitudinal cohort study of serum antibody responses towards Giardia lamblia variant-specific surface proteins in a non-endemic area.

INTRODUCTION/AIMS: The long-term humoral immune response after a natural giardiasis infection is not well understood. The aim of this study was to evaluate longitudinal serum IgA and IgG/M responses towards conserved regions of two Giardia variant-specific surface proteins (VSP) and whether these responses differ between Giardia assemblages and durations of infection. METHODS: We recruited thirty Giardia-positive patients, mainly returning travellers, and eighteen healthy adults presumed to be Giardia unexposed. Blood samples were collected before treatment, and at 6 weeks, 6 months and 12 months after the infection cleared. We used a multiplex bead-based flow cytometry immunoassay to measure Giardia specific IgA and IgG/M responses targeting two recombinant antigens from G. lamblia VSP proteins 3 and 5 (VSP3 and VSP5). RESULTS: Serum levels of anti-VSP5 and anti-VSP3 IgA decreased rapidly to low levels after treatment but continued to be substantially higher than that of presumed unexposed controls even after 6 and 12 months. The IgG/M response decreased more gradually but remained significantly higher than presumed unexposed controls at all time points, except for anti-VSP3 at 12 months. There were no significant difference in responses for infections with assemblage A and assemblage B Giardia lamblia. Chronic infections (>8 weeks) were associated with a significantly lower anti-VSP5 IgG/M response. CONCLUSION: This study describes the kinetics of the humoral immune response against two Giardia VSP proteins over one year, and the considerable cross reactivity between the two human infective Giardia assemblages. Persons with chronic Giardia infection seem to have lower levels of VSP antibodies.

Giardia lamblia: Identification of peroxisomal-like proteins.

The protozoan parasite Giardia lamblia has traditionally been reported as lacking peroxisomes, organelles involved in fatty acid metabolism and detoxification of reactive oxygen species. We here report the finding with transmission electron microscopy of an oxidase activity in cytoplasmic vesicles of trophozoites and cysts of G. lamblia. These vesicles were positive to 3,3'-diaminobenzidine and to cerium chloride staining. In addition, using bioinformatic tools, two peroxisomal proteins were identified in the G. lamblia proteome: acyl-CoA synthetase long chain family member 4 (ACSL-4) and peroxin-4 (PEX-4). With confocal and immunoelectron microscopy using polyclonal antibodies both proteins were identified in cytoplasmic vesicles of trophozoites. Altogether, our results suggest for the first time the presence of peroxisomal-like proteins in the cytoplasm of G. lamblia.

Human colostrum action against Giardia lamblia infection influenced by hormones and advanced maternal age.

Children are more susceptible to Giardia lamblia infection. Cells and hormones contained in human colostrum have an immunoprotective action against giardiasis, but the effects of advanced maternal age on these components are poorly understood. This study analyzed the colostrum of older women to determine melatonin and cortisol levels besides the participation of these hormones on the functional activity of phagocytes against G. lamblia. Colostrum samples were collected from younger (18 to 35 years old) and older (over 36 years old) lactating women. Colostrum samples were subjected to melatonin and cortisol determination, immunophenotyping, quantification of superoxide release, and assessment of phagocytic rate and microbicidal activity of phagocytes treated with hormones and in the presence of G. lamblia. Colostrum from mothers of advanced age contained higher melatonin and cortisol levels and a lower rate of cells expressing CD14+ and CD15+. In the colostru of these older mothers, melatonin increased superoxide release by phagocytes. In both groups, superoxide release by phagocytes treated with cortisol was higher in the presence of G. lamblia. In colostrum from mothers of advanced age, mononuclear (MN) phagocytes treated with melatonin showed higher phagocytosis of G. lamblia and higher microbicidal index. In younger mothers, MN and polymorphonuclear (PMN) colostrum phagocytes exhibited higher rates of G. lamblia elimination when treated with both melatonin and cortisol. In older mothers, cortisol and melatonin regulation for the functional activity of colostrum phagocytes against G. lamblia may represent an additional defense mechanism, relevant for the protection and treatment of parasitic infections in breastfed children.

Giardia lamblia: identification of molecules that contribute to direct mast cell activation.

Mast cells play a central role in the early clearance of the intestinal parasite Giardia lamblia. In a previous study, we reported that G. lamblia live trophozoites or trophozoite-derived total soluble extract induced direct activation (IgE-independent) of mast cells and release of IL-6 and TNF-α. To identify the Giardia molecules and the mast cell receptors involved in this activation, trophozoite-derived total soluble proteins separated into three fractions (F1-F3) were evaluated for its ability to activate mast cells in vitro. F2 activated mast cells in a greater extent than F1 and F3. Furthermore, F2 induced the release of IL-6 and TNF-α by mast cells. TLR2 and TLR4 expression increased slightly after mast cell stimulation with either F2 or total soluble extract; however, these receptors were not involved in F2 or total soluble extract-induced proinflammatory cytokine production. Proteins present in F2 as unique and high-intensity bands identified by liquid chromatography coupled with tandem mass spectrometry, include molecules with important biological activities such as enolase and arginine deiminase (ADI). Recombinant ADI and enolase were tested for their ability to activate mast cells, but only ADI induced a significant release of IL-6 and TNF-α. ADI product, citrulline but not ammonium, also induced mast cell release of TNF-α. Interestingly, recombinant ADI still stimulated the secretion of TNF-α by mast cells in a arginine-free medium, although in a lower extend that in the presence of arginine, indicating that either ADI itself can stimulate mast cells or through its metabolic product, citrulline.

EPIDEMIOLOGY OF CLOSTRIDIUM DIFFICILE INFECTION IN HOSPITALISED PEDIATRIC PATIENTS IN GEORGIA.

Clostridium difficile, a Gram-positive spore-forming bacillus, is the most common identifiable etiologic agent of antibiotic-associated diarrhea. The incidence of Clostridium difficile infections among hospitalized children has been increasing across the world. The aim of our study was to evaluate occurrence of Clostridium difficile and some other gastrointestinal pathogens among hospitalized pediatric patients in Georgia, as far as currently statistical data on the topic is very limited in the country. One of the objectives of the study was to test and pilot the real-time Polymerase Chain Reaction diagnostic systems for rapid and simultaneous identification of number of pathogens with a particular emphasis on diarrheal disease diagnostics as these are one of the primary public health priorities in Georgia and worldwide. Cross-Sectional study has been performed on 211 samples collected from 192 pediatric patients. Two pediatric hospitals were involved in the study: M. Iashvili Children's Central Hospital and Tbilisi Children's Clinical Hospital for Infectious Diseases. Laboratory investigations were done in the Clinic NeoLab, Tbilisi, Georgia. Study materials collected for testing were stool samples. Samples were tested by EIA kits (CerTest biotec, Zaragoza, Spain) for presence of A/B toxin according to the manufacturer's instructions. EIA test positive samples were analyzed by home-made multiplex real-time polymerase chain reaction (NeoPCR Diagnostics, NeoLab, Tbilisi, Gerogia) for confirmation of the infection and for simultaneous identification of additional gastrointestinal pathogens including Entamoeba histolitica, Giardia lamblia, Cryptsporidium parvum, Adenovirus, Rotavirus, Norovirus and Astrovirus. All samples were also tested for the presence of the above listed pathogenic agents using the same type EIA kits as for Clostridium difficile described above (CerTestbiotec, Zaragoza, Spain) for presence of the corresponding pathogen. The average age of the study participants was 3.5 years, 56.7% were male and 43.3% were female patients. Presence of Clostridium difficile have been documented in 21 samples out of 211 (10%). Besides the Clostridium difficile, other enteric pathogens have been revealed with the following frequencies: Rotavirus in 12 cases (5.7%), Adenovirus in 11 (5.2%), Giardia lamblia in 10 (4.7%), Astrovirus in 3 (1.4%), Cryptsporidium parvum in 3 (1.4%), Entamoeba histolitica in 2 (0.9%), Norovirus in 2 (0.9%). 49 samples were from out-patient cases (2 samples were positive for Clostridium difficile) and 162 samples were from in-patient cases (19 samples were positive for Clostridium difficile). Clostridium difficile is the frequent pathogenic agent causing diarrheal disease among hospitalized pediatric patients. Development of Clostridium difficile related diarrhea is associated with the antibiotic treatment of pediatric patients hospitalized due to different clinical diagnosis. Targeted early detection of these pathogens is important for the optimal management of diarrheal infection in pediatric patient which will lead to the better clinical outcome and reduction of morbidity rate among hospitalized pediatric patients.

Expression and secretion of the Giardia duodenalis variant surface protein 9B10A by transfected trophozoites causes damage to epithelial cell monolayers mediated by protease activity.

Giardia duodenalis is the protozoan parasite responsible for most cases of parasitic diarrhea worldwide. The pathogenic mechanisms of giardiasis have not yet been fully characterized. In this context parasite's excretory/secretory products have been related to the damage induced by the parasite on enterocytes. Among these is the Variable Surface Proteins (VSPs) family involved in antigenic variation and in the induction of protective response. In proteomic analyses carried out to identify the proteases with high molecular weight secreted by Giardia trophozoites during the initial phase of interaction with IEC-6 cell monolayers we identified the VSP9B10A protein. In silico bioinformatics analyses predicted a central region in residues 324-684 displaying the catalytic triad and the substrate binding pocket of cysteine proteases. The analysis of the effect of the VSP9B10A protein on epithelial cell monolayers using trophozoites that were transfected with a plasmid carrying the vsp9b10a gene sequence under the control of a constitutive promoter showed that transfected trophozoites expressing the VSP9B10A protein caused cytotoxic damages on IEC-6 and MDCK cell monolayers. This was characterized by loss of cell-cell contacts and cell detachment from the substrate while no damage was observed with trophozoites that did not express the VSP9B10A protein. The same cytotoxic effect was detected when IEC-6 cell monolayers were incubated only with supernatants from co-cultures of IEC-6 cell monolayers with VSP9B10A transfected trophozoites and this effect was not observed when transfected trophozoites were incubated with a monospecific polyclonal antibody anti-VSP9B10A previous to interaction with IEC-6 monolayers. These results demonstrate that the VSP9B10A protein secreted upon interaction with epithelial cells caused damage in these cells. Thus this protein might be considered as a conditional virulence factor candidate. To our knowledge this is the first report on the proteolytic activity from a Giardia VSP opening new research lines on these proteins.

[Giardia duodenalis and its involvement in skin diseases]. / Giardia duodenalis et son implication dans diverses dermatoses.

BACKGOUND: Over the last thirty years, the scientific community has become increasingly interested in the intestinal flora, whether commensal or pathogenic, and its impact on other organs. In dermatology, the correlation between intestinal microbial agents and cutaneous lesions is well established. Giardia duodenalis, an intestinal parasite, has been particularly widely studied. The aim of this work is to provide a review of studies demonstrating the involvement of G. duodenalis in various forms of dermatosis. PATIENTS AND METHODS: The data were obtained by an English-language literature search of Medline, PubMed and Google Scholar for the period 1975-2015. Among the thirty case reports since 1976, we selected the twenty most objective and clinically relevant. RESULTS AND DISCUSSION: This review demonstrates that intestinal giardiasis may be an etiological factor, either alone or in combination with other agents, of various dermatoses through inflammatory and allergic mechanisms or intestinal hyperpermeability. The mucocutaneous lesions are varied: urticaria, angioedema, atopic dermatitis, erythema nodosum, Wells syndrome, among others. The role and origin of the infection are often unknown, and it is thus difficult to determine the interval between parasite infestation and the onset of skin lesions. Consequently, a fecal examination to identify G. duodenalis should be considered in chronic urticaria or angioedema, and where atopic dermatitis occurs in adulthood without any specific etiology. Therapeutic test should be done in every suspicion.

The Microbiota Contributes to CD8+ T Cell Activation and Nutrient Malabsorption following Intestinal Infection with Giardia duodenalis.

Giardia duodenalis is a noninvasive luminal pathogen that impairs digestive function in its host in part by reducing intestinal disaccharidase activity. This enzyme deficiency has been shown in mice to require CD8(+) T cells. We recently showed that both host immune responses and parasite strain affected disaccharidase levels during murine giardiasis. However, high doses of antibiotics were used to facilitate infections in that study, and we therefore decided to systematically examine the effects of antibiotic use on pathogenesis and immune responses in the mouse model of giardiasis. We found that antibiotic treatment did not overtly increase the parasite burden but significantly limited the disaccharidase deficiency observed in infected mice. Moreover, while infected mice had more activated CD8(+) αß T cells in the small intestinal lamina propria, this increase was absent in antibiotic-treated mice. Infection also led to increased numbers of CD4(+) αß T cells in the lamina propria and activation of T cell receptor γδ-expressing intraepithelial lymphocytes (IEL), but these changes were not affected by antibiotics. Finally, we show that activated CD8(+) T cells express gamma interferon (IFN-γ) and granzymes but that granzymes are not required for sucrase deficiency. We conclude that CD8(+) T cells become activated in giardiasis through an antibiotic-sensitive process and contribute to reduced sucrase activity. These are the first data directly demonstrating activation of CD8(+) T cells and γδ T cells during Giardia infections. These data also demonstrate that disruption of the intestinal microbiota by antibiotic treatment prevents pathological CD8(+) T cell activation in giardiasis.

Regulation of antigenic variation in Giardia lamblia.

Antigenic variation, a clonal phenotypic variation developed by microorganisms, involves the permanent switching of homologous, antigenically different cell surface molecules. In pathogenic microorganisms, antigenic variation is often described as a mechanism to evade the host immune system and therefore is responsible for the generation of chronic and/or recurrent infections. However, antigenic variation has also been involved in expanding host diversity and differential courses of the diseases. The intestinal protozoan parasite Giardia lamblia undergoes antigenic variation through the continuous exchange of approximately 200 variant-specific surface proteins. Here we review the principal issues regarding the significance of antigenic variation during Giardia infections, the particular features of the variant-specific surface proteins, and the current knowledge on the mechanisms that regulate this process, as well as the relevance of disrupting antigenic variation as a novel approach to design effective antiparasitic vaccines.

IL-17A promotes protective IgA responses and expression of other potential effectors against the lumen-dwelling enteric parasite Giardia.

Giardia lamblia is a leading protozoan cause of diarrheal disease worldwide. It colonizes the lumen and epithelial surface of the small intestine, but does not invade the mucosa. Acute infection causes only minimal mucosal inflammation. Effective immune defenses exist, yet their identity and mechanisms remain incompletely understood. Interleukin (IL)-17A has emerged as an important cytokine involved in inflammation and antimicrobial defense against bacterial pathogens at mucosal surfaces. In this study, we demonstrate that IL-17A has a crucial function in host defense against Giardia infection. Using murine infection models with G. muris and G. lamblia, we observed marked and selective induction of intestinal IL-17A with peak expression after 2 weeks. Th17 cells in the lamina propria and innate immune cells in the epithelial compartment of the small intestine were responsible for the IL-17A response. Experiments in gene-targeted mice revealed that the cytokine, and its cognate receptor IL-17RA, were required for eradication of the parasite. The actions of the cytokine were mediated by hematopoietic cells, and were required for the transport of IgA into the intestinal lumen, since IL-17A deficiency led to marked reduction of fecal IgA levels, as well as for increased intestinal expression of several other potential effectors, including ß-defensin 1 and resistin-like molecule ß. In contrast, intestinal hypermotility, another major antigiardial defense mechanism, was not impacted by IL-17A loss. Taken together, these findings demonstrate that IL-17A and IL-17 receptor signaling are essential for intestinal defense against the important lumen-dwelling intestinal parasite Giardia.

Evaluation of ImmunoCard STAT test and ELISA versus light microscopy in diagnosis of giardiasis and cryptosporidiosis.

This study was designed to evaluate ImmunoCard STAT Cryptosporidium/Giardia rapid assay and ELISA copro-antigen assays in detecting Giardia lamblia and Cryptosporidium species in fecal samples in comparison to microscopy. Both ImmunoCard STAT and ELISA assays were evaluated with 90 stool specimens that were tested by the standard ova and parasite examination including staining with both iron hematoxylin stain and modified Ziehl Neelson stains. Counting the number of Giardia cysts and Cryptosporidia oocysts in the positive stool samples was done in order to quantify the lower limit of parasite number that was able to be detected by all included assays. Both ImmunoCard STAT and ELISA assays were compared on the basis of the attributes which are number of detected cases, sensitivity, specificity, time required for the procedure and screening, ease of performance and interpretation, and cost. Microscopic examination revealed that 13.3% of the samples were positive for Giardia and 2.2% for Cryptosporidium. By ELISA, 16.7% of the samples were infected with Giardia and 3.3% with Cryptosporidium, while by ImmunoCard STAT, 17.8 and 4.45% of the samples were positive for Giardia and Cryptosporidium, respectively. There is no statistically significant difference between the results of ELISA and ImmunoCard STAT assays. The lowest concentration detected in the stool samples was 10.50 ± 1.05 Giardia cysts and 2.83 ± 1.72 Cryptosporidium oocysts. The ImmunoCard STAT was extremely easy to read, thus requiring much less time, but its cost was much higher than ELISA. We concluded that although the overall ranking of both assays was high, the ImmunoCard STAT rapid assay was a more desirable test despite its higher cost.

Proteomic analysis in Giardia duodenalis yields insights into strain virulence and antigenic variation.

Giardia duodenalis is a protozoan parasite of the small intestine in vertebrates, including humans. Assemblage A of G. duodenalis is one of the two discrete subtypes that infects humans, and is considered a zoonotic assemblage. Two G. duodenalis Assemblage A strains BRIS/95/HEPU/2041 and BRIS/83/HEPU/106, constituting virulent and control strains respectively, were analyzed in one of the first comparative shotgun proteomic studies performed in this parasite. Protein extracts were prepared using a multiplatform approach with both an in-gel and in-solution sample preparation to enable us to assess the complementarity for future Giardia proteomic studies. Protein analysis revealed that BRIS/95/HEPU/2041 possessed a wider and more varied repertoire of variant surface proteins (VSPs), which are hypothesized to be involved in host adaptation, immune evasion, and virulence. A total of 35 VSPs were identified, with three common to both strains, six unique to BRIS/82/HEPU/106, and twenty-six unique to BRIS/95/HEPU/2041. Additionally, up to 25.6% of all differentially expressed proteins in BRIS/95/HEPU/2041 belonged to the VSP family, a trend not seen in the control BRIS/83/HEPU/106. Greater antigen variation in BRIS/95/HEPU/2041 may explain aspects of virulence phenotypes in G. duodenalis, with a highly diverse population capable of evading host immune responses.