Similarity in cyst wall protein (CWP) trafficking between encysting Giardia duodenalis trophozoites and CWP-expressing human embryonic kidney-293 cells.

Cyst wall proteins 1 and 2 (CWP1 and CWP2) are major constituents of the giardial cyst wall and are expressed with similar kinetics by encysting trophozoites. In the present study, we were interested to determine if the expression of giardial CWPs as heterologous proteins in a higher eukaryotic cell would result in their trafficking across the secretory pathway, as is the case in encysting trophozoites. Recombinant (r)CWP1 and rPro-CWP2 were detected in the lysate and culture media of transfected HEK-293 cells. We then conducted intracellular localization experiments using confocal microscopy and found that the proteins were trafficked in membrane enclosed vesicles across the secretory pathway and released to the culture medium by transfected HEK-293 cells. We then dissected the rCWP1 and rPro-CWP2 molecules to identify the portion(s) responsible for their secretion and found that the putative N-terminal signal peptide was sufficient for directing the secretion of rCWP1, while both the putative N-terminal signal peptide and the 13kDa C-terminal regions were necessary for the secretion of rPro-CWP2 by transfected HEK-293 cells. Taken together, these results demonstrate the degree of conservation of signal peptide recognition between lower and higher eukaryotes.

Oral immunization of BALB/c mice with Giardia duodenalis recombinant cyst wall protein inhibits shedding of cysts.

The process of encystation is a key step in the Giardia duodenalis life cycle that allows this intestinal protozoan to survive between hosts during person-to-person, animal-to-person, waterborne, or food-borne transmission. The release of cysts from infected persons and animals is the main contributing factor to contamination of the environment. Genes coding for cyst wall proteins (CWPs), which could be used for developing a transmission-blocking vaccine, have been cloned. Since the immunogenicity of recombinant Giardia CWP is unknown, we have investigated the immunogenicity of recombinant CWP2 (rCWP2) and its efficacy in interfering with the phenomenon of encystation taking place in the small bowels of BALB/c mice vaccinated with the recombinant protein. Here we report that the immunization of BALB/c mice with rCWP2 stimulated the immune system in a manner comparable to that for a live infection with Giardia muris cysts. Fecal and serum anti-rCWP2 immunoglobulin A (IgA) antibodies were detected in the immunized mice. In addition, anti-rCWP2 IgG1 and IgG2a antibodies were detected in the serum. mRNAs coding for Th1 and Th2 types of cytokines were detected in spleen and Peyer's patch cells from immunized mice. When the vaccinated mice were challenged with live cysts, the animals shed fewer cysts. We conclude that rCWP2 is a possible candidate antigen for the development of a transmission-blocking vaccine.

Natural transmission of Cryptosporidium parvum between dams and calves on a dairy farm.

The transmission of Cryptosporidium parvum between dams and their respective calves was studied. For this purpose, fecal specimens taken from the rectum of preparturient, parturient, and postparturient dams were analyzed for C. parvum oocysts. Fecal specimens were taken from the newborn calf 4 hr after birth. Because the environment can be a source of contamination to the animals, specimens taken from inside and outside the barn were analyzed. The sucrose concentration method together with the Zielh-Nielsen acid-fast staining method were employed to increase the chances of oocyst detection. We are reporting that at parturition, the dams shed a higher number of oocysts by comparison to the preparturient and postparturient periods. Neonates acquire the infection at birth mainly because of the high number of oocysts shed by the dams at parturition. The management practice of moving calves 4 hr after birth away from the dams and the barn reduces the number of clinical cases because they are no longer in contact with an environment that is highly contaminated. We hypothesize that the increase in the number of oocysts sheds by dams at parturition might be due to a depression of the T helper 1-type of immune response during that period.

Prevalence and geographical distribution of Giardia spp. and Cryptosporidium spp. in dairy farms in Québec.

Giardiasis and cryptosporidiosis are parasitic infections of humans, domestic animals, and wildlife. In cattle, direct transmission through the contamination of barns and/or pasture appears to be the principal mode of infection. In Canada there is only one study reported in the literature on the prevalence of giardiasis and one study on the prevalence of cryptosporidiosis. These studies were done in Alberta and Manitoba, respectively. The purpose of this study was to determine the prevalence of Giardia spp. and Cryptosporidium spp. infections in dairy farms in Québec. Calves were sampled from 505 dairy farms. We are reporting that 45.7% of the farms were found to be positive for Giardia spp. and 88.7% were infected with Cryptosporidium spp.

Exogenous cytokines released by spleen and Peyer's patch cells removed from mice infected with Giardia muris.

The role that T and B lymphocytes play in the clearance of Giardia muris in the mouse model is well known, but the cytokines produced by CD4+ T cells in response to Giardia antigenic stimulation are unknown. In this study, we have determined how Giardia trophozoite antigenic crude extract and T cell mitogens can trigger the production of cytokines by Peyer's patch and spleen cells removed from infected animals. When Giardia trophozoite proteins were used to challenge the cells in vitro, IL-4, IL-5 and IFN-gamma were not detected in the culture supernatant. When the cells were challenged with Con-A, all three cytokines were released in vitro. However, the level of each cytokine released by the spleen or Peyer's patch cells varied with the latent, acute and elimination phases of the infection. The high levels of IL-4 and IL-5 released by Peyer's patch cells confirm the importance of IgA in the control of the infection. However, we propose that the relative success of G. muris in completing its life cycle in a primary infection might be due, in part, to the stimulation of a Th2-type response (IL-4, IL-5). A stronger Th1 response (IFN-gamma) may lead to a better control of the primary infection.

Morphological changes in the jejunum of calves naturally infected with Giardia spp. and Cryptosporidium spp.

Giardiosis and cryptosporidiosis are frequently diagnosed in calves at the large animal clinic of the veterinary school. Few studies have been reported in the literature regarding pathogenesis of these two intestinal protozoa. The aims of this study were to follow the histological changes in the villi and crypts and the changes in the number of intraepithelial lymphocytes in the jejunum of naturally infected calves during the acute phase of infection. For this purpose, 29 calves aged between 7 and 10 days were bought at a local auction. The animals were housed in individual pens to avoid cross-contamination. Fecal samples were examined microscopically for the presence of Giardia cysts and Cryptosporidium oocysts, three times per week for a period of 45 days. Six calves did not pass any cysts or oocysts and were used as controls. Fifteen calves passed Giardia cysts only, five passed both cysts and oocysts, and three passed oocysts only. The villus to crypt ratio index was 1.76 in the control group and 1.08 in the Giardia-infected group. In the Cryptosporidium-infected calves, the ratio was 1.18 and calves infected with both parasites had an index of 1.37. The number of intraepithelial lymphocytes per millimeter of jejunum tissue was 21 in the control group. This number was doubled in the calves infected with Giardia, but was slightly lower in the other infected groups. All of the infected calves had intermittent diarrhea and mucus was seen in many fecal samples.

Laboratory diagnosis for Giardia lamblia infection: A comparison of microscopy, coprodiagnosis and serology.

OBJECTIVE: To evaluate newer techniques such as coproantigen detection and serology in the diagnosis of symptomatic Giardia lamblia infection. DESIGN: Blinded comparison of copro-antigen detection (by ELISA), serology (immunoglobulin IgG and IgM anti-G lamblia by ELISA, and IgG, IgM and IgA by immunoblot) and microscopy in clinical samples. Microscopic findings for three preserved stools were considered the gold standard. SETTING: Travel medicine clinic. POPULATION STUDIED: Adults, post-travel, with gastrointestinal symptomatology. MAIN RESULTS: For 152 previously collected stools, copro-antigen detection had a sensitivity of 73 of 74 (98.6%) and a specificity of 78 of 78 (100%). In clinical samples of 62 patients, eight of the 62 patients (13%) were diagnosed with G lamblia infection on microscopy. Copro-antigen diagnosis was accurate in symptomatic patients, with sensitivity of seven of eight (87.5%) and specificity of 52 of 54 (96.8%). Serology was less accurate. IgG response to G lamblia had sensitivity of four of seven and specificity of 24 of 50 (48%), and IgM response had sensitivity of three of six and specificity 27 of 48 (56%). Western blot had a sensitivity of five of seven and a specificity of 38 of 49 (78%). CONCLUSIONS: Copro-antigen diagnosis of G lamblia is highly accurate in patients with chronic gastrointestinal complaints, while serology is less accurate and appears to be less useful diagnostically.

The immune response to Giardia.

The flagellate Giardia duodenalis has been considered for many years to be a commensal living in the lumen of the small intestine of its host. It is only 25 years ago that it was accepted that Giardia is a significant pathogen of humans. Knowledge that Giardia can elicit an immune response that would probably contribute to the onset or absence of symptoms is not much older. The use of animal models to study the disease in the laboratory, together with the production of the whole life cycle in a test tube, have contributed greatly to our present knowledge of the immune responses to Giardia and of antigens that are specific to the trophozoite or cyst stages. In this review, Gaétan Faubert focuses on studies published since the last review in Parasitology Today in 1988, and examines the roles played by the humoral and cell-mediated immune responses in the control of the infection. It also covers the immunodiagnostic assays that have been recently developed on the basis of advances in our knowledge of the antigens of Giardia.

Anti-giardial activity of gastrointestinal remedies of the Luo of east Africa.

Activity in an in vitro assay with Giardia lamblia provided a test of the validity of a quantitative methodology used in an ethnobotanical survey of the Luo people of the Lake Victoria basin of Kenya and Tanzania. Forty-five taxa of remedies for gastrointestinal problems were reported by four or more independent informants and a log-linear model was used to calculate a statistical measure of informant consensus. Methanolic extracts of 21 of 36 taxa assayed were lethal or inhibited growth of Giardia trophozoites at 1000 ppm; 7 species were lethal at 500 ppm. Non-cathartic species are more likely to be active than cathartics. Lethal species of non-cathartics are reported by informants more frequently than non-lethal species although the lack of statistical significance did not provide satisfactory support for the validity of the quantitative methodology as a predictor of efficacious remedies.

Purification of a fraction of Giardia lamblia trophozoite extract associated with disaccharidase deficiencies in immune Mongolian gerbils (Meriones unguiculatus).

The effects on disaccharidase activities of challenging gerbils previously exposed to Giardia lamblia with fradions of the crude trophozoite extract were examined. Gel filtration of the soluble extract on a Sephacryl S-200 HR column resulted in 3 fradions: F1, F2 and F3. Only a challenge with fraction F1 (0.1 mg total dose) was found to induce disaccharidase deficiencies. Boiling F1 prior to challenge did not change this effect on the enzyme activities. However, the decreases were not obtained when the total F1 dose was reduced to 0.05 mg. Column chromatography of fraction F1 under dissociating and reducing conditions resulted in 2 further fractions: F1a and F1b. Challenging immune gerbils with F1b led to impairments of disaccharidose activity similar to those obtained with F1. Protein analysis of the crude extract, as well as the fractions of the extract, revealed several high and low molecular weight bonds. These findings indicate that a constituent(s) of fraction F1b is the portion of the parasite which induces disaccharidase deficiencies in immune gerbils. This fraction consists of proteins ranging in molecular weight from 32 to 200 kDa. In addition the G. lamblia fraction involved in the decreases in enzyme activity is heat-stable.

Disaccharidase deficiencies in Mongolian gerbils (Meriones unguiculatus) protected against Giardia lamblia.

The activities of the disaccharidases lactase, maltase, sucrase and trehalase were examined in gerbils during Giardia lamblia infections. In a primary infection with trophozoites, the activities of all four enzymes were reduced from day 10 post-infection (p.i.) and remained at low levels well past the elimination phase of the infection. However, during a challenge infection, the disaccharidase decreases were short-lived, with impairments being seen only on days 2 and/or 4 post-challenge (p.c.). Sucrase activity was not affected by a challenge infection. When 0.1 mg of a soluble extract of G. lamblia trophozoites was used to challenge gerbils previously exposed to the live parasite, the pattern and duration of enzyme deficiencies were comparable with those observed after the challenge with the live parasite. In addition, decreasing the extract dose used to challenge the gerbils led to smaller disaccharidase deficiencies. G. lamblia-infected gerbils were also challenged with a soluble extract of Entamoeba histolytica trophozoites, and this had no effect on the disaccharidase activities. Therefore, the presence of the intact parasite was not necessary to induce enzyme reductions in immune animals. In addition, the effects seen during the secondary infection were parasite-specific and may have involved the host's immune response to Giardia antigens. Immune gerbils were further challenged with the in vitro-released excretory/secretory products of G. lamblia. Under our experimental conditions, disaccharidase activities were found to be affected by these products in a manner that was inconsistent with the results of the live parasite challenge, and this merits further study.

Giardia lamblia: traffic of a trophozoite variant surface protein and a major cyst wall epitope during growth, encystation, and antigenic switching.

Both trophozoites and cysts of Giardia lamblia have unique outer surfaces that protect them from very different hostile environments. However, little is known about the transport of these important molecules to the cell surface. We used monospecific anti-recombinant TSA 417 antibodies and mAb 8C5 in double label immunoelectron microscopy to compare the localization and transport of this major trophozoite surface antigen (TSA) with that of a prominent cyst wall epitope during vegetative growth, encystation, and antigenic switching in vitro. TSA 417 is a marker of the constitutive transport of the major plasma membrane protein, while the 8C5 epitope traces a differentiation-regulated secretory pathway to the cyst wall. Both proteins localized to the nuclear envelope endoplasmic reticulum (ER) cisternae, ER, and cytoplasmic membrane cisternae, reflecting their site of synthesis, but only the differentiation-specific epitope 8C5 localized to the encystation-specific vesicles (ESV). These large secretory vesicles form only during encystation and transport cyst antigens to the nascent wall. In contrast, only TSA 417 was found on the outer surface of the plasmalemma of trophozoites and encysting cells and underlaying the walls of many cysts, while only 8C5 localized to the cyst wall. As encystation progressed, TSA 417 disappeared from the plasmalemma and increased in the lysosome-like PV and other large cytoplasmic vesicles. In contrast to their segregation in the ESV and on the cell surface, both TSA 417 and 8C5 were found in the peripheral vesicles, presumably an endocytic compartment. We propose that this may be the initiation of a stage in differentiation-driven antigenic switching of TSA 417, in which this antigen is no longer synthesized or exported to the plasmalemma, but is taken back inside the cell.

Recognition of Giardia lamblia cyst-specific antigens by monoclonal antibodies.

Immunization of BALB/c mice with a sonicated extract of in vitro-generated Giardia lamblia cysts produced six cyst-specific monoclonal antibodies (MoAbs). Two MoAbs (8C5.C11 and 5A4.G6), which recognize proteinaceous cyst antigens, were selected for further study. In indirect immunofluorescence (IFA), MoAb 8C5.C11 reacted with encystation-specific vesicles in trophozoites beginning 3 h after the induction of encystation in vitro. This MoAb also recognized cysts which began to appear at 12 h. In contrast, MoAb 5A4.G6 stained only cyst walls. In Western blots, both MoAbs also reacted with cyst antigens, but not trophozoite antigens. MoAb 8C5.C11 first recognized cyst antigen from 3 h encysting cultures, reacting with 26, 28, 42 and 46 kD bands. MoAb 5A4.G6 reacted with a 38 kD band, beginning with 12 h encysting cultures. When added to G. lamblia encysting cultures before the appearance of cysts (0 to 9 h) and in the presence of a source of complement, MoAb 8C5.C11 caused a significant reduction in the numbers of water-resistant cysts produced in vitro compared to the control. MoAb 5A4.G6 did not affect in vitro encystation. These findings confirm the heterogeneity of cyst antigens, and also indicate that the process of encystation in vitro can be interrupted by antibodies and complement.

Antimicrobial action of antibodies against Giardia muris trophozoites.

The activities of immune serum and trophozoite-specific MoAb were examined in vitro and in vivo. Immune serum and anti-Giardia muris MoAb caused immobilization of the trophozoites in vitro and were cytotoxic for trophozoites in the presence of exogenous complement. Both immune serum obtained from experimentally infected mice and anti-G. muris MoAb administered directly into the duodenum of mice significantly reduced the number of trophozoites in the small intestine during the acute phase of the infection. These results suggest that serum antibodies play a central role in the elimination of the primary Giardia infection.

Interaction of mebendazole with tubulin from body wall muscle, intestine, and reproductive system of Ascaris suum.

The binding of tritiated mebendazole, a benzimidazole anthelmintic, to tubulin derived from intestine, body wall muscle, and reproductive system of adult Ascaris suum was examined and compared. Mebendazole binding was resolved into specific and nonspecific binding and the binding affinity (Ka) and maximum binding at infinite ligand concentration (Bmax) determined. Electron microscopy was performed to assess the tubulin in various tissues of A. suum quantitatively by observing the presence of microtubules. Total binding was highest in intestine followed by body wall muscle. It was least in the reproductive system. The intestine demonstrated greater specific binding per milligram of protein than the body wall muscle. However, in the reproductive system extract, high affinity binding was not detected. After correction for nonspecific binding of ligand, the results indicated that the Bmax of mebendazole for the tubulin of A. suum intestine was about 3-fold higher than for that of body wall muscle. The Ka of mebendazole for intestinal tubulin was similar to that for body wall muscle. Electron microscopy of A. suum tissues demonstrated that the tubulin content decreased from the intestine through the body wall muscle to the reproductive system. Differences in tubulin content from different tissues may determine the selective sensitivity of these tissues to benzimidazole attack.

Comparative studies on Giardia lamblia encystation in vitro and in vivo.

The distribution of Giardia lamblia (WB strain) trophozoites, encysting trophozoites (i.e., trophozoites bearing encystation-specific vesicles [ESV]) and cysts in the intestines of gerbils was examined over a period of 30 days. Trophozoites and encysting trophozoites were found in 3 equal length sections of the small intestine and in much lower numbers in the colon. Cysts were consistently found only in the second and third sections of the small intestine and in the colon. In a comparative encystation study in vitro, 4 populations of WB strain trophozoites were used: WB (uncloned), D1 (a recent clone of WB), WB-C6 (a 1983 clone of WB), and V1 (a recent subclone of WB-C6). The WB-C6 and V1 populations produced significantly higher numbers of encysting trophozoites and cysts in vitro than did the WB and D1 populations. In addition, WB-C6 and V1 produced significantly more water-resistant cysts in vitro than WB and D1. However, there was no difference in fecal cyst excretion in gerbils infected with the 4 populations of WB-derived trophozoites. One cycle of encystation/excystation in gerbils increased the in vitro encystation of WB and D1 populations but decreased in vitro encystation of C6 and V1. We could detect no difference in protein profiles or isoenzyme patterns between these 4 populations of cells before or after passage in gerbils.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization and biological activities of anti-Brugia pahangi tubulin monoclonal antibodies.

Three monoclonal antibodies (mAb) specific to beta-tubulin were used to investigate the heterogeneity of tubulins from nematodes and mammals. Western blot analysis of one-dimensional SDS-PAGE showed that anti-Brugia pahangi tubulin mAb 1B6 and P3D react with epitope(s) specific to nematode beta-tubulin and recognize tubulin from adults and microfilariae of B. pahangi, adult B. malayi and Dirofilaria immitis, eggs of Haemonchus contortus and adult Ascaris suum. However, the same mAb did not recognize tubulin from trophozoites of Giardia lamblia, pig brain or 3T3 mouse fibroblast cells. In two-dimensional SDS-PAGE, mAb 1B6 recognized one isoform of beta-tubulin and mAb P3D recognized two beta-tubulin isoforms. Limited proteolysis showed that mAb 1B6 reacted with the amino-terminal fragments of beta-tubulin. In contrast, mAb P3D recognized the carboxy-terminal fragments of beta-tubulin. In ELISA, mAb P3D reacted with an 18 amino acid peptide corresponding to residues 430-448 of B. pahangi beta-tubulin. These observations confirm that the epitope of mAb P3D is located on the extreme carboxy-terminal region. Immunogold labelling of adult B. pahangi sections with mAb P3D revealed the presence of beta-tubulin isoforms in the cuticle, hypodermal layer and somatic muscle blocks of B. pahangi. Under in vitro conditions, mAb P3D caused 80% reduction in worm viability, during exposure over 48 h.

Identification of tubulin isoforms in different tissues of Ascaris suum using anti-tubulin monoclonal antibodies.

Three monoclonal antibodies specific to alpha- and beta-tubulin were used to examine the expression of tubulin isoforms in the intestine, reproductive tract and body wall muscle of A. suum. The tubulins were found to be different in their isoelectric points, number of isoforms and peptide maps with Western blot analysis of one-dimensional polyacrylamide gel confirming the presence of alpha-, beta 1- and beta 2-tubulin. Commercial cross-reactive anti-alpha and anti-beta MAbs 356 and 357 recognized tubulin from A. suum tissues as well as from pig brain, whereas anti-A. suum beta-tubulin specific MAb P3D6 recognized tubulin from the A. suum tissues only. Two-dimensional gel analysis showed different isoform patterns in different A. suum tissues with anti-A. suum beta-tubulin MAb P3D6 and cross-reactive beta-tubulin MAb 357 recognizing 2-4 beta-tubulin isoforms and anti-alpha-tubulin MAb 356 recognizing 1-6 alpha-tubulin isoforms. Different peptide maps of tubulin were observed in the three tissues, when subjected to limited proteolysis followed by SDS-PAGE. The data indicate that different tubulins are found in different tissues of adult A. suum.