Giardia muris and Giardia duodenalis groups: ultrastructural differences between the trophozoites

Trophozoites of the Giardia muris group from hamsters, domestic rats and mice and of the Giardia duodenalis group from hamsters and domestic rats were examined by transmission electron microscopy. The basic ultrastructure of the trophozoites was similar. Differences were shown in the morphology of the ventrolateral flange of the trophozoites of Giardia muris and Giardia duodenalis groups. Marginal plates are less developed in the species of the Giardia duodenalis group. In this group, the distal extremity of the lateral flange is short and thick and the marginal plate does not penetrate into the distal extremity of the flange. In the Giardia muris group, the ventro-lateral flange is well developed and narrow and the marginal plate penetrates the distal extremity of the flange. The osmiophilic lamella, which accompanies the dorsal surface of the marginal plate is seen only in the Giardia muris group.

Avaliação de massas específicas de cistos de Giardia duodenalis e Entamoeba coli

Cistos de Giardia duodenalis e Entamoeba coli foram observados quanto à flutuação em soluções de sacarose de massas específicas diversas (1.040, 1.050, 1.060, 1.070,1.080,1.090,1.100, 1.150,1.200 e 1.250 kg/m³), contidas em câmaras de contagem de 0,17 mm de altura. Contaram-se os cistos que flutuaram e os que sedimentaram tendo sido calculadas as porcentagens respectivas. As diferenças de flutuabilidade dos cistos de cada uma das espécies não foram consideráveis. Soluções de massa específica igual a 1.200 kg/m³ fizeram flutuar 88,49% dos cistos de G. duodenalis e 95,71% dos de E. coli. Os maiores valores de flutuabilidade estiveram associados à massa específica 1.250 kg/m³ e foram, respectivamente, 89,15% e 98,59% para cistos de G. duodenalis e de E. coli.

Giardia duodenalis: INTER-STRAIN VARIABILITY OF PROTEINS, ANTIGENS, PROTEASES, ISOENZYMES AND NUCLEIC ACIDS

Giardia duodenalis isolates from asymptomatic or symptomatic patients and from animals present similarities and differences in the protein composition, antigenic profile, pattern of proteases and isoenzymes, as well as in nucleic acids analysis. In the present overview, these differences and similarities are reviewed with emphasis in the host-parasite interplay and possible mechanisms of virulence of the protozoon.

Analysis of proteins from membrane and soluble fractions of Giardia duodenalis trophozoites of two Brazilian axenic strains

In the present study, we have analyzed by sodium docecyl sulphate - polyacrilamide gel electrophoresis (SDS-PAGE), immunoblotting and Concanavalin A blotting (Con A blotting) proteins of membrane fractions and soluble fractions obtained from Giardia duodenalis trophozoites of two axenic strains isolated in Brazil from a symptomatic (BTU-11) and an asymptomatic patient (BTU-10), as compared to the reference strain Portland 1. Both Brazilian strains showed a complex and homogeneous electrophoretic pattern of proteins, but some differences could be observed. Several glycoproteins were detected, particularly the proteins of 81, 72, 59 kDa and the protein of 62 kDa in the membrane proteins and cytosol, respectively. Many antigenic components were revealed by anti-Giardia rabbit IgG antibodies in the immunoblotting analysis. Among these components, the membrane protein of 32 kDa and the cytosol protein of 30 kDa could be related to giardin, as previously demonstrated.

Occurrence of enteroparasites in day care centers in Botucatu (São Paulo State, Brazil) with emphasis on Cryptosporidium sp., Giardia duodenalis and Enterobius vermicularis

The occurrence of the enteroparasites was verified in 279 children (0 to 6 years) of four municipal day cares of Botucatu/SP. Three samples of each child's feces were collected and processed by the methods of Hoffman-Pons-Janner, Faust and Ritchie and subsequent coloration of the fecal smear by the methods of Auramina-O and Ziehl-Neelsen modified for diagnosis of Cryptosporidium sp. and Graham method for diagnosis of Enterobius vermicularis. Of the analyzed children we verified a prevalence of intestinal parasitism in 53.40%, and the most frequent parasite was Giardia duodenalis (26.88%). Significant association was verified among enteroparasitosis, family income, maternal education and age; the lowest enteroparasite frequency occurred in children of families with larger income and higher education. It was observed that G. duodenalis is more prevalent in children from 0 to 4 years and E. vermicularis is more frequent in children between three and four years old. The high enteroparasite prevalence in day cares suggests complex structure in its epidemiology, where factors beyond sanitation should be considered.

Partial characterization of proteolytic activity in Giardia duodenalis purified proteins

This report describes a preliminary characterization of proteolytic activity of proteins isolated from lysate of Giardia trophozoites of an axenic Brazilian strain. Fractions obtained by high-performance liquid chromatography (FPLC) were tested in SDS-polyacrylamide gel for the protein profiles, and the proteases activity was analyzed using gelatin impregnated SDS-PAGE. The proteases characterization was based on inhibition assays employing synthetic inhibitors for cysteine (E-64, IAA), serine (PMSF, TPCK, TLCK, and elastatinal), metalo (EDTA) and aspartic (pepstatin) proteases. Among thirty eluted fractions, polypeptide bands were observed in eight of them, however, proteolytic activity was detected in four ones (F23, F24, F25 and F26). Protein profiles of these fractions showed a banding pattern composed by few bands distributed in the migration region of 45 to < 18 kDa. The zymograms revealed proteolytic activity in all the four fractions assayed, mainly distributed in the migration region of 62 to 35 kDa. Among the profiles, the main pronounced zones of proteolysis were distinguished at 62, 55, 53, 50, 46 and 40 kDa. In inhibition assays, the protease activities were significantly inhibited by cysteine (E-64) and serine proteases (TPCK, TLCK and elastatinal) inhibitors. Gels incubated with other cysteine and serine protease inhibitors, IAA and PMSF, respectively, showed a decrease in the intensity of hydrolysis zones. Indeed, in the assays with the inhibitors EDTA for metalloproteases and pepstatin for aspartic proteases, none inhibition was detected against the substrate. These observations are relevants, especially if we consider that to define the real role of the proteases in host-parasite interaction, the purification of these enzymes for detailed studies may be warranted.

Classic and molecular study of Giardia duodenalis in children from a daycare center in the region of Presidente Prudente, São Paulo, Brazil

Epidemiological studies on giardiasis by using molecular techniques such as RAPD (Randomly Amplified Polymorphic DNA) may give information on factors related to the transmission of Giardia duodenalis. The aim of this work was to assess the epidemiology of G. duodenalis in 101 children attended at a daycare center in Presidente Bernardes, SP, Brazil. After parasitological examinations in feces samples, 15 children presented cysts of G. duodenalis. Their respective parents, brothers and pets, besides the daycare center workers, also had their feces submitted to parasitological analysis. Seven mothers and nine brothers also presented G. duodenalis cysts, while fathers, daycare workers and pets (dogs) did not presented the parasite. Besides the 15 cases with G. duodenalis, other 23 children presented other enteroparasites (Entamoeba coli, Endolimax nana, Enterobius vermicularis, Ascaris lumbricoides and Trichuris trichiura). Samples of G. duodenalis cysts from children and their relatives were submitted to molecular typing by RAPD after genomic DNA extraction and amplification of a fragment of the 18S rDNA region by PCR. After examining 31 isolates of G. duodenalis (children and their respective mothers and brothers), it was concluded that the parasite transmission occurred in children, probably during daily cohabitation at the daycare center, but not at home among their relatives or pets.

Giardia duodenalis: genotypic comparison between a human and a canine isolates

INTRODUCTION: Evidence suggests that giardiasis is a zoonotic disease. The present work aimed to evaluate the genetic identity of Giardia duodenalis isolated from human and dog fecal samples from Belo Horizonte. METHODS: Human and dog fecal samples were cultured for isolation of G. duodenalis. To determine the genotype of the isolates, primers that amplify a specific region in rRNA of the protozoan were used. RESULTS: Two G. duodenalis isolates were obtained, which belong to the subgroup A genotype. CONCLUSIONS: These findings suggest that the transmission of giardiasis follows a zoonotic pattern.

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

Throphozoites of Giardia duodenalis group obtained from fragments or scratched of hamster's mucosa were examined by transission electron microscopy. The fine structure of the trophozoites are presented and comapred 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 crossbridge 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.

Influence of Refrigeration and Formalin on the Floatability of Giardia duodenalis Cysts

Giardia duodenalis cysts obtained from fresh fecal samples, fecal samples kept under refrigeration and fecal samples treated with formalin were studied as to their floatability on sucrose solutions with the following specific gravities: 1,040 kg/m3; 1,050 kg/m3; 1,060 kg/m3; 1,070 kg/m3; 1,080 kg/m3; 1,090 kg/m3; 1,100 kgm3; 1,150 kg/m3; 1,200 kg/m3; and 1,250 kg/m3, contained within counting-chambers 0.17 mm high. Cysts that floated on and those settled down as sediments were counted, and had their percentages estimated. Sucrose solutions of 1,200 kg/m3 specific gravity (the average specific gravity of diluting liquids employed in floatation techniques) caused to float 77.7%, 78.4% and 6.6% of the G. duodenalis cysts obtained, respectively, from fresh fecal samples, fecal samples kept under refrigeration, and fecal samples treated with formalin. Cysts obtained both from fresh fecal samples and fecal samples kept under refrigeration presented similar results concerning floatability. It was observed, however, that the treatment of feces with formalin diminished the cysts floatability under the various specific gravities studied. This results should influence, the recommendations for transport and storage of fecal samples used for parasitological coproscopy.

Detection of Giardia duodenalis antigen in human fecal eluates by enzyme-linked immunosorbent assay using polyclonal antibodies

The present study developed and standardized an enzime-linked immunosorbent assay (ELISA) to detect Giardia antigen in feces using rabbit polyclonal antibodies. Giardia cysts were purified from human fecal samples by sucrose and percoll gradients. Gerbils (Meriones unguiculatus) were infected to obtain trophozoites. Rabbits were inoculated with either cyst or trophozoite antigens of 14 Colombian Giardia isolates to develop antibodies against the respective stages. The IgG anti-Giardia were purified by sequential caprylic acid and ammonium sulfate precipitation. A portion of these polyclonal antibodies was linked to alkaline phosphatase (conjugate). One hundred and ninety six samples of human feces, from different patients, were tested by parasitologic diagnosis: 69 were positive for Giardia cysts, 56 had no Giardia parasites, and 71 revealed parasites other than Giardia. The optimal concentration of polyclonal antibodies for antigen capture was 40 µg/ml and the optimal conjugate dilution was 1:100. The absorbance cut-off value was 0.24. The parameters of the ELISA test for Giardia antigen detection were: sensitivity, 100% (95% CI: 93.4-100%); specificity, 95% (95% CI: 88.6-97.6%); positive predictive value, 91% (95% CI: 81.4-95.9%); and negative predictive value, 100% (95% CI: 96.1-100%). This ELISA will improve the diagnosis of Giardia infections in Colombia and will be useful in following patients after treatment.

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.

Giardia duodenalis: analysis of secreted proteases upon trophozoite-epithelial cell interaction in vitro

Protease secretion by Giardia duodenalis trophozoites upon interaction with epithelial cells and its association with the parasite adhesion was studied in co-cultures of parasites with IEC6 epithelial cell monolayers in the presence or absence of protease inhibitors. Proteolytic activity in supernatants from trophozoites was enhanced when they were co-cultured with IEC6 cells. This activity was strongly inhibited by pre-incubation of live trophozoites with E-64 and TPCK and a concomitant inhibition of parasite adhesion to IEC6 cells was observed. These data suggest that trophozoites secrete cysteine-type proteases that play a role in the adhesion of G. duodenalis to epithelial cells.