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.     

A Three Nucleotide Signature Sequence in Small Subunit rRNA Divides Human Giardia in Two Different Genotypes

ABSTRACT. The nucleotide sequence of the 16S rRNA gene, part of the 23S rRNA gene and the spacer DNA region was determined for Giardia duodenalis , obtained from humans in The Netherlands (AMC-4) and Washington State (CM). These rDNA sequences differ from other G. duodenalis isolates (Portland-1 and BRIS/83/HEPU/106) both of which have virtually identical rDNA sequences. The most characteristic feature was found close to the 5'end of the 16S rRNA. The Portland-1 - Bris/83/HEPU/106 type has GCG in position 22–24, while AMC-4 and CM have AUC in this position. These two sequences, present in an otherwise conserved region of the 16S rRNA, are "signature" sequences, which divide Giardia isolates into two different groups.     

Mixed Giardia duodenalis assemblage A and E infections in calves

A molecular epidemiological study was conducted on 100 dairy (499 calves) and 50 beef (333 calves) farms in Belgium to estimate the prevalence of different Giardia duodenalis assemblages in calves younger than 10 weeks of age. Positive samples from the epidemiological study and from a previous clinical study were selected and genotyped based on the amplification of the beta-giardin gene. To investigate the occurence of mixed assemblage A and E infections in calves, a novel assemblage-specific PCR was developed based on the triose-phosphate isomerase gene. The prevalence was 22% (95% Probability Interval (PI): 12-34%) in dairy calves and 45% (95% PI: 30-64%) in beef calves. In total, 120 Giardia-positive samples from dairy and beef calves collected in the epidemiological study and from clinically affected calves were identified based on the amplification of the beta-giardin gene. Overall G. duodenalis assemblage E was more prevalent (in 64% of the samples), although the majority (59%) of the dairy calves were infected with G. duodenalis assemblage A. Furthermore, mixed G. duodenalis assemblage A and E infections were identified in 31% of the calf samples (n=101) using the assemblage-specific PCR. We believe this is the first report of mixed infections in calves, and the results of the present study indicate that calves, although mainly infected with the host-specific G. duodenalis assemblage E, are frequently infected with the zoonotic assemblage A, either as a mixed or mono-infection, suggesting that calves might be underestimated as a potential zoonotic reservoir for human infections.     

Antigenic analysis of Giardia duodenalis strains isolated in Alberta

Giardia duodenalis is a common intestinal parasite in most parts of the world. In Canada it is associated with both endemic and epidemic infections that are often transmitted by the waterborne route. Although G. duodenalis strains have been isolated from several animals, the role of other mammals in human infection is unclear. We have isolated and cultured G. duodenalis trophozoites from domestic and wild animals in Alberta and compared them with a human isolate by protein gel electrophoresis and immunoblot analysis. All strains examined share a similar polypeptide profile and important protein antigens. Prominent antigens of 62, 52, 38, and 31 kilodaltons are conserved. The 52- and 31-kilodalton proteins are the major surface-exposed trophozoite components. The high degree of antigenic sharing among strains from different hosts suggests that there may be a wide range of potential reservoirs for G. duodenalis infections.     

Virulent Avian Giardia duodenalis Pathogenic for Mice

Early in 1995, a sulphur-crested cockatoo captured in the wild died along with several other cage mates, apparently of an overwhelming, acute infection of Giardia. Trophozoites isolated from the dead bird and established in traditional Giardia axenic medium were infective to mice and established chronic infections associated with weight gain impairment. Genetically and morphologically, the Giardia isolated from the bird belonged to the duodenalis group. Here, Jacqui Upcroft, Ann McDonnell and Peter Upcroft present data on pathogenic avian Giardia with he potential to contaminate watersheds and discuss the implications.     

Identification of cyst and trophozoite antigens from Colombian Giardia duodenalis isolates recognized by IgA

Little is known about the role of IgA in the immune response against Giardia duodenalis infection. The current study identified the antigens of Colombian G. duodenalis isolates which stimulate the production of IgA anti-G. dudoenalis. Cyst and trophozoite stage proteins were separated by SDS-PAGE and their antigenicity was determined by Western blot. Without 2-mercapto ethanol (2-ME), the protein profile of the cyst stage showed 24 proteins within a molecular weight range of 23-270 kDa; with 2-ME, 35 polypeptides ranging from 22 to 241 kDa were distinguished. The trophozoite stage protein profile without 2-ME was formed by 16 proteins within the range of 24-270 kDa; with 2-ME, 45 proteins were present between 18 and 241 kDa. The identification of 20 and 29 antigens from the cyst and trophozoite stage, respectively, suggested that G. duodenalis stimulates a specific humoral immune response in the human host. The antigens of 31, 57, 110, 133, and 170 kDa recognized by anti-G duodenalis IgA in both cysts and trophozoites corresponded with G. duodenalis isolates from other geographic regions, whereas those of 35, 38, 43, 45, 49, 52, 60, 62, 65, 72, 82, 99, 145, 155, and 185 kDa seemed specific to Colombian isolates. This indicated that antigens of 57, 65, 145, and 170 kDa, recognized by anti-G. duodenalis IgA antibodies in cysts (with frequencies between 82% and 96%) and trophozoites (with frequencies between 86% and 97%) can be considered identification markers for G. duodenalis infections.     

Dientamoeba fragilis is more prevalent than Giardia duodenalis in children and adults attending a day care centre in Central Italy

Giardia duodenalis is a well recognised enteropathogen, while Dientamoeba fragilis is rarely detected and consequently it is not recognised as an important human pathogen. In 2002-2003, a survey has been carried out on enteroparasites in faecal samples of outpatients attending a day care centre in the town of Perugia (Central Italy). To improve the detection level, at least three samples from each patient were collected at different days and within two hours from defecation. The coproparasitological examination has been carried out by direct microscopic examination, faecal concentration, and Giemsa and modified Ziehl-Nielsen stainings of faecal smears. The genotypes of Giardia duodenalis isolates were determined by PCR of the beta-giardin gene. Of 1,989 enrolled people (966 children, 1,023 adults), 165 persons (8.3%; 153 adults, 15.0%; 12 children, 1.2%), were positive for parasites, but only 1 12 adults (73.2% of those infected) and eight children (66.7% of those infected) harboured D. fragilis and G. duodenalis. Both the Assemblages A and B were detected in 18 G. duodenalis isolates examined at the beta-giardin gene. The higher prevalence of D. fragilis infections than that of G. duodenalis is probably related to the method used, a procedure, which is rarely followed in laboratories for the diagnosis of enteric parasites. These epidemiological data suggest that when faecal samples are examined after a period of time and without Giemsa staining, most D. fragilis infections goes undetected.     

Human giardiasis: genotype linked differences in clinical symptomatology

Giardia duodenalis infection in humans can cause a variety of clinical symptoms. The relation between clinical symptomatology and the Giardia isolate genotype was studied in 18 Dutch patients infected with G. duodenalis who visited their general practitioner. Contrary to earlier studies, a 100% correlation between severity of diarrhoeal complaints and genotype was found: assemblage A isolates were solely detected in patients with intermittent diarrhoeal complaints, while assemblage B isolates were present in patients with persistent diarrhoeal complaints. These results are significant because they show for the first time that genetically linked features of G. duodenalis are major determinants in the severity of infection in human giardiasis.     

Host immunity and pathogen strain contribute to intestinal disaccharidase impairment following gut infection

Infection or other inflammatory insults in the small intestine often result in reduced disaccharidase enzyme levels. Using a mouse model of giardiasis, we examined the role of host immunity and pathogen virulence in mediating disaccharidase deficiency postinfection (p.i.). C57BL/6J mice were infected with two strains, WB and GS, of the human parasite Giardia duodenalis. The levels of sucrase, maltase, and lactase decreased in wild-type mice p.i. with the GS strain but not with the WB strain. Both CD4-deficient and SCID mice failed to eliminate the infection and did not exhibit disaccharidase deficiency. β(2)-Microglobulin knockout animals controlled infections similar to wild-type mice but exhibited no decrease in disaccharidase activity. Analysis of cytokine production by spleen and mesenteric lymph node cells showed production of IL-4, IL-10, IL-13, IL-17, IL-22, TNF-α, and IFN-γ p.i. with both WB and GS, with IFN-γ being the dominant cytokine for both parasite strains. Mesenteric lymph node cells produced lower levels of cytokines compared with splenocytes in response to parasite extract, although the overall pattern was similar. These data suggest that T cell responses mediate parasite clearance whereas also contributing to pathogenesis. They also demonstrate that differences in pathogen strain can also determine the outcome of infection and further our understanding of the clinical variation seen in human giardiasis.     

Rapid DNA extraction methods and new primers for randomly amplified polymorphic DNA analysis of Giardia duodenalis.

A randomly amplified polymorphic DNA (RAPD) procedure using simple genomic DNA preparation methods and newly designed primers was optimized for analyzing Giardia duodenalis strains. Genomic DNA was extracted from in vitro cultivated trophozoites by five freezing-thawing cycles or by sonic treatment. Compared to a conventional method involving proteinase K digestion and phenol extraction, both freezing-thawing and sonication were equally efficient, yet with the advantage of being much less time- and labor-intensive. Five of the 10 tested RAPD primers produced reproducible polymorphisms among five human origin G. duodenalis strains, and grouping of these strains based on RAPD profiles was in agreement among these primers. The consistent classification of two standard laboratory reference strains, Portland-1 and WB, in the same group confirmed previous results using other fingerprinting methods, indicating that the reported simple DNA extraction methods and the selected primers are useful in RAPD for molecular characterization of G. duodenalis strains.     

Excystation and culturing of human and animal Giardia spp. by using gerbils and TYI-S-33 medium

Mongolian gerbils were used as an animal model to excyst and host Giardia spp. isolated from meadow voles, dogs, beavers, and humans. Both cysts and trophozoites were used to establish infections. Gerbils were infected with Giardia duodenalis from beaver, dog, and human sources, and the trophozoites were extracted and cultured in Diamond TYI-S-33 medium. The use of gentamicin and ampicillin in the medium, coupled with treatment of gerbils with gentamicin before they were sacrificed, permitted the elimination of trophozoite purification techniques before culturing. An extract of whole bovine calf blood, CLEX, was substituted for fetal bovine serum in TYI-S-33 medium and was found to be both adequate and less expensive.     

Excystation and culturing of human and animal Giardia spp. by using gerbils and TYI-S-33 medium.

Mongolian gerbils were used as an animal model to excyst and host Giardia spp. isolated from meadow voles, dogs, beavers, and humans. Both cysts and trophozoites were used to establish infections. Gerbils were infected with Giardia duodenalis from beaver, dog, and human sources, and the trophozoites were extracted and cultured in Diamond TYI-S-33 medium. The use of gentamicin and ampicillin in the medium, coupled with treatment of gerbils with gentamicin before they were sacrificed, permitted the elimination of trophozoite purification techniques before culturing. An extract of whole bovine calf blood, CLEX, was substituted for fetal bovine serum in TYI-S-33 medium and was found to be both adequate and less expensive.     

Protease activity in Giardia duodenalis trophozoites of axenic strains isolated from symptomatic and asymptomatic patients

We have examined by gelatin-SDS-PAGE the protease activity in cell lysates of Giardia duodenalis trophozoites of two axenic strains isolated in Brazil from a symptomatic patient (BTU-11) and an asymptomatic carrier (BTU-10), and the reference strain Portland 1 (P1). The proteolysis band patterns showed differences among strains isolated from asymptomatic and symptomatic individuals. The lysate of the strain BTU-10, showed only five hydrolysis bands, while a greater number of bands (10-11 bands) was seen in strains BTU-11 and P1. The protease activity in all lysates was inhibited by cysteine (E-64 and iodoacetamide) and serine proteases (TPCK and TLCK) inhibitors, but not by PMSF and EDTA. In general, the results revealed protease activities in G. duodenalis trophozoites of Brazilian axenic strains and the predominance of cysteine proteinases. It should be stressed the inter-strain difference in hydrolysis band patterns observed between strains isolated from symptomatic patients and the strain obtained from an asymptomatic carrier.     

Serial subcultivation of Giardia lamblia in Keister's modified TYI-S-33 medium containing ultroser G.

The ability of Giardia strains of the duodenalis type to grow in Keister's modified TYI-S-33 medium varies with serum lot. Recently, strains of Giardia including MR4, WB, and Human-1-Portland, have been cultivated in Keister's modified TYI-S-33 medium containing the serum substitute Ultroser G and have been cultured serially as least 40 times. An optimal concentration of 8% Ultroser G promotes maximal growth in Keister's modified TYI-S-33 medium for all three strains. This concentration of Ultroser G will produce a two-log increase in the number of trophozoites in approximately three days post-inoculation. Generation times for the trophozoites ranging from 6 to 11 h have been achieved in Keister's modified TYI-S-33 containing 10% adult bovine serum and from 8 to 13 h in Keister's modified TYI-S-33 with 8% Ultroser G. Despite the excellent growth of Giardia strains in medium containing Ultroser G, the maximum trophozoite density is only about half of that achieved in Keister's modified TYI-S-33 medium supplemented with 10% adult bovine serum. Comparisons of trophozoites grown with serum or the serum substitute reveal no discernable differences in morphology and motility. Additionally, these strains have been successfully cryopreserved and revived in Keister's modified TYI-S-33 medium supplemented with Ultroser G. Because Ultroser G is a characterized mixture of six main groups of ingredients (growth factors, adhesion factors, mineral trace elements, hormones, binding proteins, and vitamins), the variability in cell proliferation that may occur when changing serum lots should be minimized when using this product.     

Giardia duodenalis and Cryptosporidium spp. in the intestinal contents of ringed seals (Phoca hispida) and bearded seals (Erignathus barbatus) in Nunavik, Quebec, Canada

The prevalence of Giardia duodenalis and Cryptosporidium spp. was determined for ringed and bearded seals harvested for food in the Nunavik region in northern Quebec, Canada. Flow cytometric results demonstrated that G. duodenalis was present in the intestinal contents of 80% of the ringed seals and 75% of the bearded seals tested, while Cryptosporidium spp. were present in 9% of the ringed seals and none of the bearded seals. Prevalence of both parasites was highest in animals less than 1 yr of age. Giardia sp. isolates from ringed seals were identified as G. duodenalis Assemblage B, which is commonly identified in human infections. The high prevalence of G. duodenalis in ringed seals, and the presence of Assemblage B in these animals, highlights the potential for zoonotic transmission to the Inuit people, who consume dried seal intestines and uncooked seal meat.     

Giardia sp.: comparison of electrophoretic karyotypes

Species in the genus Giardia have been named on the basis of host specificity, cell dimensions, and median body morphology. Despite these criteria, the species taxonomy of Giardia is still in question. To investigate Giardia taxonomy on a molecular level, Giardia chromosomal DNA was analyzed by orthogonal-field-alternation gel electrophoresis (OFAGE) and transverse alternating field electrophoresis (TAFE). Chromosomal DNA of G. duodenalis isolates (human, muskrat, sheep, dog, beaver), G. muris (mouse), and G. ardeae (great blue heron) were subjected to OFAGE and TAFE analyses. Comparable DNA patterns were obtained by both electrophoretic methods, but OFAGE required 8 days while TAFE required only 3 days. DNA patterns among all G. duodenalis isolates, although quite similar to each other, were distinctly different from those of G. muris and G. ardeae; G. muris and G. ardeae DNA patterns were distinctly different from each other. A G. duodenalis (Portland 1) total DNA probe hybridized to the DNA of all G. duodenalis isolates on Southern blots, but not detectably to G. muris and G. ardeae DNA. Similarly, G. muris and G. ardeae total DNA probes only hybridized detectably to their respective DNA. One probe that appears to hybridize to the DNA of all G. duodenalis and to G. ardeae DNA rather than G. muris DNA has been developed. Another probe that hybridizes only to G. muris and G. ardeae DNA has been developed. These data suggest that the differentiation of Giardia isolated from host and environmental samples may eventually be accomplished by DNA probes. Additionally, these techniques perhaps combined with other criteria may lead to the establishment of a sound taxonomic scheme for this genus.     

Serial Subcultivation of Giardia lamblia in Keister's Modified TYI-S-33 Medium Containing Ultroser G

ABSTRACT. The ability of Giardia strains of the duodenalis type to grow in Keister's modified TYI-S-33 medium varies with serum lot. Recently, strains of Giardia including MR4, WB, and Human-1-Portland, have been cultivated in Keister's modified TYI-S-33 medium containing the serum substitute Ultroser G and have been cultured serially at least 40 times. An optimal concentration of 8% Ultroser G promotes maximal growth in Keister's modified TYI-S-33 medium for all three strains. This concentration of Ultroser G will produce a two-log increase in the number of trophozoites in approximately three days post-inoculation. Generation times for the trophozoites ranging from 6 to 11 h have been achieved in Keister's modified TYI-S-33 containing 10% adult bovine serum and from 8 to 13 h in Keister's modified TYI-S-33 with 8% Ultroser G. Despite the excellent growth of Giardia strains in medium containing Ultroser G, the maximum trophozoite density is only about half of that achieved in Keister's modified TYI-S-33 medium supplemented with 10% adult bovine serum. Comparisons of trophozoites grown with serum or the serum substitute reveal no discernable differences in morphology and motility. Additionally, these strains have been successfully cryopreserved and revived in Keister's modified TYI-S-33 medium supplemented with Ultroser G. Because Ultroser G is a characterized mixture of six main groups of ingredients (growth factors, adhesion factors, mineral trace elements, hormones, binding proteins, and vitamins), the variability in cell proliferation that may occur when changing serum lots should be minimized when using this product.     

Axenic culture and characterization of Giardia ardeae from the great blue heron (Ardea herodias)

Trophozoites of Giardia ardeae were obtained from the great blue heron (Ardea herodias) and established in axenic culture using the TYI-S-33 medium. The generation time in culture for G. ardeae was 22-25 hr, which was 3-fold longer than for Giardia duodenalis (WB strain). A morphological comparison of trophozoites in the original intestinal isolate to those grown in culture revealed that they were identical for the following characteristics: a pyriform-shaped body, a ventral adhesive disc with a deep notch in the posterior border, teardrop-shaped nuclei, pleomorphism in median body structure ranging from a round-oval appearance (Giardia muris type) to that of a clawhammer (G. duodenalis type), and a single caudal flagellum on the right side (as viewed dorsally) with the left one being rudimentary. Analysis of the chromosomal migration patterns was performed by orthogonal-field-alternation gel electrophoresis and demonstrated that the pattern for G. ardeae was distinctly different from that for G. duodenalis (Portland 1-CCW strain). Bacterial symbionts were seen attached to trophozoites in the original isolate but could not be detected in cultured trophozoites using scanning electron microscopy, fluorescence light microscopy using the Hoechst 33258 dye for DNA localization, or by standard microbiological techniques using nonselective media for growing aerobic or anaerobic bacteria. This study demonstrated that avian-derived Giardia could be grown in axenic culture; based on morphological criteria and chromosomal migration patterns, that G. ardeae should be considered a distinct species; and that rationale for determining Giardia spp., based on median body structure alone, should no longer be considered adequate for classification at the species level.     

Intestinal IgA responses to Giardia muris in mice depleted of helper T lymphocytes and in immunocompetent mice

Immunocompetent mice infected with Giardia muris generate an intestinal antibody response to this parasite and clear G. muris infection. Previous work has shown that G. muris infection is prolonged in mice that have been depleted of helper (CD4+) T lymphocytes by treatment with a monoclonal antibody (mAb) directed against the murine CD4 antigen. The aim of the present study was to compare the intestinal anti-Giardia antibody response in immunocompetent mice and in mice depleted of helper T (Th) lymphocytes by treatment with anti-CD4 mAb. Immunocompetent mice generated an IgA response to G. muris, as judged by the presence of IgA on Giardia trophozoites harvested from the intestine of these animals more than 10 days after the start of the infection. The anti-Giardia IgA response was impaired in mice depleted of Th lymphocytes, as judged by virtual absence of immunofluorescent staining of trophozoites from these animals for surface-bound IgA. Clearance of G. muris infection was impaired by treatment of mice with anti-CD4 mAb. The results suggest that Th (CD4+) lymphocytes are important for the generation of a local IgA response against G. muris trophozoites in the mouse intestine and that IgA anti-trophozoite antibody may contribute to the clearance of G. muris from the intestine of immunocompetent mice.     

Oral vaccination of mice against Helicobacter pylori with recombinant Lactococcus lactis expressing urease subunit B

To determine whether a protective immune response could be elicited by oral delivery of a recombinant live bacterial vaccine, Helicobacter pylori urease subunit B (UreB) was expressed for extracellular expression in food-grade bacterium Lactococcus lactis . The UreB-producing strains were then administered orally to mice, and the immune response to UreB was examined. Orally vaccinated mice produced a significant UreB-specific serum immunoglobulin G (IgG) response. Specific anti-UreB IgA responses could be detected in the feces of mice immunized with the secreting lactococcal strain. Mice vaccinated orally were significantly protected against gastric Helicobacter infection following a challenge with H. pylori strain SS1. In conclusion, mucosal vaccination with L. lactis expressing UreB produced serum IgG and UreB-specific fecal IgA, and prevented gastric infection with H. pylori .