Identification of antigens of Colombian Giardia duodenalis isolates recognized by total IgG and subclasses

Antigen profiles were described for Giardia duodenalis cysts and trophozoites that are recognized by IgG and its anti-G. dudodenalis subclasses (IgG1, IgG2, IgG3, IgG4). Antigens were identified by Western blot from G. duodenalis cyst and trophozoite isolates. Cysts and trophozoites were each subjected to protein separation by SDS-PAGE. The proteins were then transferred to nitrocellulose membranes by electroimmunoblot, and their antigenicity was determined by exposing them to sera from patients with confirmed diagnosis of G. duodenalis infection. The antigen-antibody reaction was revealed by specific alkaline phosphatase antibody conjugates against IgG, IgG2, IgG3, IgG4: bands were visualized by addition of the substrate 5-bromo-4-chloro-3-indolyl-phosphate and the stain nitro blue tetrazolium. The bands were read and analyzed by linear regression using Quantity One software. Thirty two antigens were simultaneously recognized by total IgG anti-G. duodenalis in the cyst and trophozoite stages. The antigens varied in molecular weight from 22 to 185 kDa. Nineteen antigens were identified by both IgG, and IgG3 anti-G duodenalis, with molecular weights ranging from 42 to 180 kDa. IgG2 and IgG4 did not identify any antigen in either stage. The antigens of molecular weights 27, 30, 31, 33, 45, 49, 57, 78, 89 and 170 kDa are shared with G. duodenalis isolates from other geographical regions of Colombia. The recognition of cyst and trophozoite antigens of Colombian G. duodenalis isolates by IgG, IgG1 and IgG3 anti-G. duodenalis suggested that they are involved in the induction of the host immune response.     

Prevalence and genotyping of human isolates of Giardia duodenalis from Albania

Microscopical and PCR-based techniques were performed in order to investigate the prevalence of infection and the genotypes of Giardia duodenalis from 125 stool samples collected from children living in the urban and the rural areas of Tirana (Albania) and hospitalized with acute gastroenteritis. 7 out of 125 samples resulted positive for Giardia at the microscopic examination (5.6%). In 50 selected samples including the 7 samples positive for Giardia by microscopy, 3 and 15 additional positive samples were detected by immunofluorescence and PCR, respectively. Seasonality appeared as an important parameter to be evaluated in order to better understand the prevalence of infection. Sequence analysis revealed both human Assemblage A and B. This result represents the first data on G. duodenalis genotypes in Albania.     

Immunoblot patterns of Giardia duodenalis isolates from different hosts and geographical locations

Eighteen isolates of Giardia duodenalis from animal and human sources were studied for protein differences by polyacrylamide gel electrophoresis and for antigenic differences by immunoblot analysis. The polyacrylamide gels showed that whilst the isolates were for the most part homogeneous in their protein banding patterns, some isolates did show some differences. The immunoblot analysis yielded many bands, including prominent bands of 32 and 66 kilodaltons. Five of the six isolates that showed differences in protein banding pattern also showed differences in antigenic reactivity. Our findings suggest that differences can be seen with the use of immunoblotting and that this technique is a tool that may be useful for isolate differentiation when used in conjunction with other techniques.     

Improvement in cyst recovery and molecular detection of Giardia duodenalis from stool samples

Molecular Biology Reports - Molecular detection of Giardia duodenalis by polymerase chain reaction (PCR) is difficult in faecal samples due to inhibitors that contaminate DNA preparations, or due...     

Identification of nucleoli in the early branching protist Giardia duodenalis

Giardia duodenalis has been described as 'anucleolated'. In this work we analysed the subcellular distribution of several nucleolar markers in Giardia nuclei using silver and immunostaining techniques for electron and confocal laser microscopy as well as expression of epitope-tagged proteins in transgenic trophozoites. We identified anteronuclear fibrogranular structures corresponding to nucleolar organising regions with recruited ribonucleoprotein complexes, rRNA and epitope-tagged fibrillarin and rRNA-pseudouridine synthase (CBF5). Recombinant fibrillarin and CBF5 were targeted to this subcompartment. This study demonstrates the presence of nucleoli in G. duodenalis and provides a model to analyse minimal requirements for nucleolar assembly and maintenance in eukaryotic cells.     

High-resolution electron microscopic evidence for the filamentous structure of the cyst wall in Giardia muris and Giardia duodenalis

High-resolution morphological studies of the cyst wall of Giardia spp. were performed using low-voltage scanning electron microscopy (LVSEM) and transmission electron microscopy (TEM). The cyst wall was composed of membranous and filamentous layers. The membranous layer consisted of an inner and an outer cyst membrane separated by a thin layer of cytoplasm. The filamentous layer contained individual filaments that ranged from 7 to 20 nm in diameter when measured by LVSEM, formed a dense meshwork with branches or interconnections, and were occasionally arranged on the surface in whorled patterns. Cysts of Giardia muris from mice, Giardia duodenalis from dogs, pigs, voles, beavers, muskrats, and humans, and Giardia psittaci from a bird (parakeet), possessed an essentially identical wall composed of filaments. Inducement of excystation in viable Giardia cysts produced a dramatic increase in the interfilament spacing over an entire cyst, but none was observed in heat-killed or chemically fixed control cysts. These results demonstrated that the cyst wall of Giardia spp. was composed of a complex arrangement of filaments, presumably formed during the process of encystment.     

Occurrence and Molecular Identification of Giardia duodenalis from Stray Cats in Guangzhou,Southern China

The objective of this study was to genetically characterize isolates of Giardia duodenalis and to determine if zoonotic potential of G. duodenalis could be found in stray cats from urban and suburban environments in Guangzhou, China. Among 102 fresh fecal samples of stray cats, 30 samples were collected in Baiyun district (urban) and 72 in Conghua district (suburban). G. duodenalis specimens were examined using light microscopy, then the positive specimens were subjected to PCR amplification and subsequent sequencing at 4 loci such as glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), β-giardin (bg), and small subunit ribosomal RNA (18S rRNA) genes. The phylogenetic trees were constructed using obtained sequences by MEGA5.2 software. Results show that 9.8% (10/102) feline fecal samples were found to be positive by microscopy, 10% (3/30) in Baiyun district and 9.7% (7/72) in Conghua district. Among the 10 positive samples, 9 were single infection (8 isolates, assemblage A; 1 isolate, assemblage F) and 1 sample was mixed infection with assemblages A and C. Based on tpi, gdh, and bg genes, all sequences of assemblage A showed complete homology with AI except for 1 isolate (CHC83). These findings not only confirmed the occurrence of G. duodenalis in stray cats, but also showed that zoonotic assemblage A was found for the first time in stray cats living in urban and suburban environments in China.     

Limitations of tpi and bg genes sub-genotyping for characterization of human Giardia duodenalis isolates

The intestinal protozoan Giardia duodenalis includes 2 genetically distinct assemblages, A and B, which are responsible for human infections. Little is known so far on the genotypes of G. duodenalis human isolates in France. The present characterization of 19 French clinical isolates was aimed at determining their genotype patterns and associations with clinical symptoms, and in vivo metronidazole resistance, respectively. Based on both triose-phosphate isomerase (tpi) and β-giardin (bg) gene sequences, twelve isolates were identified as assemblage A, and 7 as assemblage B for the 2 gene loci. Sub-genotyping heterogeneities were observed in 15/19 isolates attributed to either A or B assemblage. They include frequent mismatches and intra-assemblage discordances and mixed positions, which were found more frequently in tpi than in bg sequences, and in assemblage B than in assemblage A sequences. No association was found between sub-genotypes, clinical symptoms and metronidazole sensitivity. Present data underline the need for improvements in the standardization of G. duodenalis multilocus genotyping approach for further molecular epidemiologic studies of giardiasis.     

Identification of developmentally regulated Giardia lamblia cyst antigens using GCSA-1, a cyst-specific monoclonal antibody

GCSA-1, a monoclonal antibody raised against cysts generated in vitro was shown to be Giardia cyst-specific by immunoblot analysis and immunofluorescence. GCSA-1 recognized four polypeptides ranging from 29-45 kD present in the cyst wall. These antigens appeared within eight hours of exposure of trophozoites to encystation medium and were shown to be synthesized by encysting parasites by means of metabolic labelling with [35S]-cysteine. Trophozoites were not stained by the antibody. GCSA-1 also reacted with in vivo cysts obtained from faeces of infected humans, gerbils and mice. These data demonstrate that the determinants recognized by GCSA-1 are early cyst antigens which are developmentally regulated and conserved components of the cyst wall. The actual role of the antigens detected by GCSA-1 in encystation are unknown, but they represent a potential target for strategies directed at inhibiting this process.     

A simple method for cloning Giardia duodenalis from cultures and fecal samples

Using a novel method for cloning Giardia duodenalis from cultures and fecal samples, 47 clones from 7 isolates were established in vitro. Average colony-forming efficiency in established cultures was 43.2% compared to 11.2% when cloning directly from excystation. The highest success rate of cloning was found with the Portland (P1, ATCC No. 30888) isolate, with a colony-forming efficiency of 92.7%. Cloned and parent populations were compared over a range of 13 enzymes using starch gel electrophoresis. No genetic difference was found between any of the clones and the parent isolates.     

Genetic Exchange Within and Between Assemblages of Giardia duodenalis

ABSTRACT. Meiotic sex evolved early in the history of eukaryotes. Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis ), a parasitic protist belonging to an early diverging lineage of eukaryotes, shows no cytological or physiological evidence of meiotic or sexual processes. Recent molecular analyses challenge the idea that G. duodenalis is a strictly clonal organism by providing evidence of recombination between homologous chromosomes within one subgroup (Assemblage A) of this species as well as genetic transfer from one subgroup to another (Assemblage A–B). Because recombination is not well documented and because it is not known whether the observed inter-assemblage transfer represents true reciprocal genetic exchange or a non-sexual process, we analyzed genic sequences from all major subgroups (Assemblages A–G) of this species. For all assemblages, we detected molecular signatures consistent with meiotic sex or genetic exchange, including low levels of heterozygosity, as indicated by allelic sequence divergence within isolates, and intra- and inter-assemblage recombination. The identification of recombination between assemblages suggests a shared gene pool and calls into question whether it is appropriate to divide the genetically distinct assemblages of G. duodenalis into a species complex.     

Comparison of Giardia lamblia and Giardia muris cyst inactivation by ozone.

Inactivation of Giardia lamblia and Giardia muris cysts was compared by using an ozone demand-free 0.05 M phosphate buffer in bench-scale batch reactors at 22 degrees C. Ozone was added to each trial from a concentrated stock solution for contact times of 2 and 5 min. The viability of the control and treated cysts was evaluated by using the C3H/HeN mouse and Mongolian gerbil models for G. muris and G. lamblia, respectively. The resistance of G. lamblia to ozone was not significantly different from that of G. muris under the study conditions, contrary to previously reported data that suggested G. lamblia was significantly more sensitive to ozone than G. muris was. The simple Ct value for 2 log unit inactivation of G. lamblia was 2.4 times higher than the Ct value recommended by the Surface Water Treatment Rule.     

Comparative studies on the growth dynamics of two genetically distinct isolates of Giardia duodenalis in vitro.

The in vitro growth behaviour of the intestinal protozoan Giardia duodenalis was studied in detail and comparisons were made between two genetically and biologically distinct cloned isolates. Replicates of each clone were grown at six different initial cell concentrations and in culture media at four different pH values. Significant differences in in vitro growth were found between the two isolates, BAH12 and P1. BAH12 had a specific narrow pH requirement, with satisfactory growth only obtained at pH 6. The mean generation time of BAH12 at pH 6 between days 1 and 3 was 10.8 h, compared to an average of 6 h for the same period for P1, both at pH 6 and pH 7. Comparative health of cultures was assessed during both the pH and growth experiments using a suite of six variables. Consistent changes in the health of cultures over time were found to reflect growth behaviour over time. These results provide the first detailed evidence that genetically different isolates of Giardia may differ in such fundamental biological parameters as growth rate and pH requirements. These differences may have important epidemiological and taxonomic implications.     

Phosphoglycerate kinase and fructose bisphosphate aldolase of Candida albicans as new antigens recognized by human salivary IgA

BackgroundCandida albicans is an opportunistic dimorphic fungus commonly present in the human oral cavity that causes infections in immunocompromised patients. The antigen variability, influenced by growth conditions, is a pathogenicity factor.AimsTo determine the effect of nutritional and heat stress on the antigen expression of C. albicans, and to identify major antigens recognized by human salivary secretory immunoglobulin A (sIgA).MethodsUnder various different nutritional conditions, heat shock was induced in C. albicans cells in stationary and exponential growth phases. The expression of protein determinants of C. albicans was assessed by Western blot analysis against human saliva. The antigens were purified and characterized by two-dimensional electrophoresis and identified by protein microsequencing.ResultsFive antigens recognized by salivary IgA were characterized as mannoproteins due to their reactivity with concanavalin A. They did not show reactivity with anti-heat shock protein monoclonal antibodies. Two of them (42 and 36 kDa) were found to be regulated by heat shock and by nutritional stress and they were identified as phosphoglycerate kinase and fructose bisphosphate aldolase, respectively.ConclusionsThese glycolytic enzymes are major antigens of C. albicans, and their differential expression and recognition by the mucosal immune response system could be involved in protection against oral infection.     

Identification of Giardia lamblia isolates susceptible and resistant to infection by the double-stranded RNA virus

The presence or absence of the Giardia lamblia double-stranded RNA virus (GLV) was surveyed among 38 axenic isolates of G. lamblia derived from both humans and animals. Of the 28 isolates lacking the virus, 19 could readily be infected by the virus. The remaining 9 isolates proved to be resistant to GLV infection even when the ratio between virus to parasite reached as high as 10(6) to 1. Evidence is also presented indicating that there are at least two "Portland 1" strains being used by the current scientific community, one containing the virus and the other lacking the virus.     

Ultrastructure of Giardia duodenalis trophozoites group from hamster: some relevant aspects.

Trophozoites of Giardia duodenalis group obtained from fragments or scratched of hamster's mucosa were examined by transmission electron microscopy. The fine structure of the trophozoites are presented and compared with those described for other animals. Some of the trophozoites present the cytoplasm full of glycogen, rough endoplasmic reticulum-like structures and homogeneous inclusions not enclosed by membranes, recognized as lipid drops, which had not been observed in Giardia from other animals. The adhesive disk is composed of a layer of microtubules, from which fibrous ribbons extend into the cytoplasm; these ribbons are linked by layer of cross-bridge filaments that shows an intermediary dense band, described for the first time in this paper. The authors regard this band as the result of the cross-bridge filaments slinding in the medium region between adjacent fibrous ribbons, and suggest a contractile activity for them. The role of the adhesive disk on the trophozoite mechanism of attachment to host mucosa is also discussed.