Cross-species transmission of Giardia spp.: inoculation of beavers and muskrats with cysts of human, beaver, mouse, and muskrat origin

Giardia cysts isolated from humans, beavers, mice, and muskrats were tested in cross-species transmission experiments for their ability to infect either beavers or muskrats. Giardia cysts, derived from multiple symptomatic human donors and used for inoculation of beavers or muskrats, were shown to be viable by incorporation of fluorogenic dyes, excystation, and their ability to produce infections in the Mongolian gerbil model. Inoculation of beavers with 5 x 10(5) Giardia lamblia cysts resulted in the infection of 75% of the animals (n = 8), as judged by the presence of fecal cysts or intestinal trophozoites at necropsy. The mean prepatent period was 13.1 days. An infective dose experiment, using 5 x 10(1) to 5 x 10(5) viable G. lamblia cysts collected by fluorescence-activated cell sorting, demonstrated that doses of between, less than 50, and less than 500 viable cysts were required to produce infection in beavers. Scanning electron microscopy of beaver small intestine revealed that attachment of G. lamblia trophozoites produced lesions in the microvillous border. Inoculation of muskrats with G. lamblia cysts produced infections when the dose of cysts was equal to or greater than 1.25 x 10(5). The inoculation of beavers with Giardia ondatrae or Giardia muris cysts did not produce any infection; however, the administration to muskrats of Giardia cysts of beaver origin resulted in the infection of 62% of the animals (n = 8), with a prepatent period of 5 days. Our results demonstrated that beavers and muskrats could be infected with Giardia cysts derived from humans, but only by using large numbers of cysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cross-species transmission of Giardia spp.: inoculation of beavers and muskrats with cysts of human, beaver, mouse, and muskrat origin.

Giardia cysts isolated from humans, beavers, mice, and muskrats were tested in cross-species transmission experiments for their ability to infect either beavers or muskrats. Giardia cysts, derived from multiple symptomatic human donors and used for inoculation of beavers or muskrats, were shown to be viable by incorporation of fluorogenic dyes, excystation, and their ability to produce infections in the Mongolian gerbil model. Inoculation of beavers with 5 x 10(5) Giardia lamblia cysts resulted in the infection of 75% of the animals (n = 8), as judged by the presence of fecal cysts or intestinal trophozoites at necropsy. The mean prepatent period was 13.1 days. An infective dose experiment, using 5 x 10(1) to 5 x 10(5) viable G. lamblia cysts collected by fluorescence-activated cell sorting, demonstrated that doses of between, less than 50, and less than 500 viable cysts were required to produce infection in beavers. Scanning electron microscopy of beaver small intestine revealed that attachment of G. lamblia trophozoites produced lesions in the microvillous border. Inoculation of muskrats with G. lamblia cysts produced infections when the dose of cysts was equal to or greater than 1.25 x 10(5). The inoculation of beavers with Giardia ondatrae or Giardia muris cysts did not produce any infection; however, the administration to muskrats of Giardia cysts of beaver origin resulted in the infection of 62% of the animals (n = 8), with a prepatent period of 5 days. Our results demonstrated that beavers and muskrats could be infected with Giardia cysts derived from humans, but only by using large numbers of cysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multinucleate cyst formation in Acetabularia mediterranea after x-irradiation

Cells of Acetabularia mediterranea were irradiated with increasing doses of X-rays (64.5–258·10^-4 kC kg^-1). The cells are radioresistant up to 193.5·10^-4 kC kg^-1 in terms of growth and progression through he life cycle but the morphogenesis of whorls, caps, and cysts is accompanied by morphological alterations. Microscopical examination of cyst bearing caps in irradiated cells has shown the presence of giant cysts neighboring particularly small ones. Photographic recording of cyst development showed that the multinucleate cap cytoplasm partitions into multinucleate portions rather than uninucleate ones as in the control cells. After complete cleavage a cyst wall is deposited onto the multinucleate cytoplasm. In contrast to uninucleate cysts with one lid the wall contains multiple lids. Their number appears to correspond to the number of nuclei in the cytoplasm compartment during cleavage. The results indicate that X-rays preferentially inhibit the synthesis of a factor which plays a role in establishing the normal spatial morphogenetic pattern necessary for cyst formation.     

Reduction in fecal excretion of Giardia cysts: effect of cholestasis and diet

Bile is a major growth factor for the proliferation of Giardia spp. trophozoites in the small intestine and, at high concentrations, stimulates encystment of trophozoites. This report demonstrates that surgical cholestasis to interrupt the flow of bile from liver to intestine or the use of bile-binding resins in the diet can both dramatically decrease the fecal excretion of Giardia muris cysts. Cholestasis produced a 3 log reduction in excretion of G. muris cysts within 24 hr of surgery and a 4 log reduction after 3 days. Sham controls showed no difference in cyst excretion from presurgical control values. Two isocaloric diets were studied: a control diet (N) of Purina mouse chow containing 5% celufil and an experimental diet (CR) containing 5% cholestyramine, a resin that binds bile. Compared with the N diet, the CR diet was associated with reductions in cyst excretion of 3 logs within 1 day. Despite lowered excretion of G. muris cysts in mice fed the cholestyramine diet, the trophozoite recovery from the duodenum was similar with both diets. Cyclic feeding of the CR diet and the N diet at 3-day intervals produced significant oscillations (changes of 3-4 logs) in fecal cyst shedding. The significant reductions in fecal excretion of cysts observed with agents that bind bile suggests that diets capable of binding bile might be a therapeutic means to minimize the fecal excretion of cysts and thereby may help to reduce the risk of spreading giardiasis through fecal-oral contamination.     

Ultrastructural Evidence for the Presence of Bacteria,Viral-like Particles,and Mycoplasma-like Organisms Associated with Giardia spp.1

ABSTRACT. Giardia trophozoites and cysts, isolated from mammalian and avian hosts, were examined by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and by fluorescent light microscopy for the presence of microbial symbionts. Mycoplasma-like organisms were observed on the surfaces of trophozoites isolated from the prairie vole, laboratory rat, and beaver. Intracellular bacteria were observed by TEM in the trophozoites and cysts of G. microti and by fluorescence microscopy in trophozoites and cysts of Giardia spp. isolated from beaver, muskrat, great-blue heron, and the green heron. Trophozoites of G. muris from rat small intestine contained viral-like particles measuring 60 nm in diameter. These observations suggest that biological associations between Giardia spp. and diverse microbes may be more common than formerly appreciated. It also raises the possibility of transmission of these apparent symbionts, via the Giardia cyst, to other mammalian hosts including man.     

Giardia duodenalis: Kinetics of cyst elimination and the systemic humoral and intestinal secretory immune responses in gerbils (Meriones unguiculatus) experimentally infected

This study aimed to determine the pre-patent period and to evaluate the kinetics of cyst elimination and the systemic humoral (IgA, IgG₁, IgG_2a, IgM, IgE) and intestinal secretory (IgA) immune responses in gerbils (Meriones unguiculatus) experimentally innoculated with different doses of Giardia duodenalis trophozoites. Forty-eight animals aged 6-8 weeks were used, equally distributed among six groups, five groups innoculated with different doses of trophozoites (10¹, 10², 10³, 10⁴, 10⁵) and one control (non-infected) group. Coproparasitological examinations were carried out daily up to 91 days after inoculation (d.a.i.) to determine the pre-patent period and the kinetics of cyst elimination. Blood and stool samples were weekly collected for antibody assays. The pre-patent period was observed from the 9 d.a.i. onwards, with intermittent elimination of variable quantities of cysts up to 27 d.a.i.. All infected gerbils, irrespective of the dose received, were able to mount systemic humoral immune responses as evidenced by specific IgM titers from 7 to 28 d.a.i., corresponding to the peak of cyst elimination, followed by high and persistent IgG1 titers. Intestinal secretory responses were also seen with two peaks of fecal IgA titers, corresponding to IgM and IgG1response peaks, respectively. In conclusion, systemic and intestinal humoral immune responses were related to the control of giardiasis in this experimental model.     

Effect of Phorbol Myristate Acetate,a Tumor-Promoting Agent,on the Growth of Mycobacterium lepraemurium in the Mouse Footpad

Phorbol myristate acetate (PMA), a potent inflammatory agent with tumor-promoting activity, was examined for its effect on the growth of Mycobacterium lepraemurium (MLM) in the left hind footpad of mice. When the animals were infected with 10⁴ MLM and received multiple injections of 3 μg of PMA in the infection site weekly during the first 2 months and biweekly thereafter, the growth of the bacilli was markedly enhanced. PMA injection in the infection site resulted in severe footpad swelling accompanied by inflammatory signs such as redness, edema, induration, and sometimes ulcer. Acetic acid, as potent an inflammatory and hyperplastic agent as PMA but without any appreciable tumor-promoting action, did not stimulate MLM growth when it was injected biweekly in the site of infection with MLM at a dose of 30 μmol per injection. When mice were infected with 10⁸ MLM, proper elimination of bacilli from the infection site was observed during the first 3 months. In this case, multiple injections of PMA in the infection site resulted in the enhancement of the elimination of MLM by host defense mechanisms, although PMA caused as severe inflammation as that observed when MLM infection was produced with a small inoculum (10⁴ MLM). In both cases, dexamethasone was synergistic with, but indomethacin and L -1-tosylamide-2-phenyl-ethylchloromethyl ketone were antagonistic to, the effect of PMA.     

Ultrastructural evidence for the presence of bacteria, viral-like particles, and mycoplasma-like organisms associated with Giardia spp

Giardia trophozoites and cysts, isolated from mammalian and avian hosts, were examined by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and by fluorescent light microscopy for the presence of microbial symbionts. Mycoplasma-like organisms were observed on the surfaces of trophozoites isolated from the prairie vole, laboratory rat, and beaver. Intracellular bacteria were observed by TEM in the trophozoites and cysts of G. microti and by fluorescence microscopy in trophozoites and cysts of Giardia spp. isolated from beaver, muskrat, great-blue heron, and the green heron. Trophozoites of G. muris from rat small intestine contained viral-like particles measuring 60 nm in diameter. These observations suggest that biological associations between Giardia spp. and diverse microbes may be more common than formerly appreciated. It also raises the possibility of transmission of these apparent symbionts, via the Giardia cyst, to other mammalian hosts including man.     

Giardia duodenalis: pathological alterations in gerbils, Meriones unguiculatus, infected with different dosages of trophozoites

To examine the infection kinetics and development of alterations in the small intestine of gerbils (Meriones unguiculatus), 72 gerbils were divided into six groups (A to F), with A serving as control and the others inoculated with increasing doses of trophozoites from Giardia duodenalis human isolate. The infection kinetics and the development of histopathological alterations were monitored by optical scanning electron microscopy (SEM). A 12-day prepatent period was observed, with intermittent elimination up to day 35 after inoculation. Statistically significant differences were found between the mean number of trophozoites recovered, per group, on the days of sacrifice, and a positive correlation between the inoculum dosage and the number of trophozoites recovered. Morphometrically, the villus:crypt ratio showed a drop in all the groups when compared with the control group. SEM revealed an increase in mucus production in the inoculated animals and the presence of trophozoite clusters at the top and base of the villi. The dosage of trophozoite inoculum does not interfere in the ability for infection to occur or in the development of histopathological alterations generated by intestinal colonization.     

Inducible nitric oxide synthase in innate immune cells is important for restricting cyst formation of Toxoplasma gondii in the brain but not required for the protective immune process to remove the cysts

Significantly larger numbers of Toxoplasma gondii cysts were detected in the brains of RAG1^?/?NOS2^?/? than RAG1^?/? mice following infection. In contrast, the cyst numbers markedly decreased in a same manner in both strains of mice after receiving CD8⁺ immune T cells. Thus, NOS2-mediated innate immunity is important for inhibiting formation of cysts in the brain but not required for the T cell-initiated cyst removal, which is associated with phagocyte accumulation. Treatment with chloroquine, an inhibitor of endolysosomal acidification, partially but significantly inhibited the T cell-mediated cyst removal, suggesting that phagosome–lysosome fusion could be involved in the T. gondii cyst elimination.     

Oxygen Uptake In Cysts and Trophozoites of Giardia Lamblia

ABSTRACT. Oxygen uptake in cysts and trophozoites of the parasitic protozoan Giardia lamblia was examined. Both showed oxygen uptake activity, but that of cysts was only 10% to 20% that of trophozoites. Oxygen dependence of oxygen uptake in cysts and trophozoites showed oxygen maxima above which oxygen uptake decreased. the oxygen concentration at which the oxygen uptake rate was greatest was higher for trophozoites than for cysts. the effect of various inhibitors on cyst and trophozoithe oxygen uptake suggested that flavoproteins and quinones play some role in oxygen uptake. the substrate specificities and the effect of inhibitors on G. lamblia trophozoites were similar to those observed for G. muris. Metronidazole, the drug most commonly used in treatment of giardiasis, inhibited oxygen uptake and motility in trophozoites; however, it had no obvious effect on either oxygen uptake or excystation in cysts. Menadione, a redox cycling naphthaquinone, first stimulated, then completely inhibited, oxygen uptake in cysts and trophozoites; a complete loss of cyst viability and trophozoite motility was also observed. the effect of menadione on G. Iamblia may indicate that redox cycling compounds have potential as chemotherapeutic agents for the treatment of giardiasis.     

A comparison of the Giardia lamblia trophozoite and cyst transcriptome using microarrays

Background  

Compared with many protists, Giardia lamblia has a simple life cycle alternating between cyst and trophozoite. Most research on the molecular biology of Giardia parasites has focused on trophozoites and the processes of excystation and encystation, whereas cysts have attracted less interest. The striking morphological differences between the dormant cyst and the rapidly dividing and motile trophozoite implies profound changes in the metabolism as the parasite encysts in the host's intestine and excysts upon ingestion by a new host.     

Changes in the N-glycome, glycoproteins with Asn-linked glycans, of Giardia lamblia with differentiation from trophozoites to cysts

Giardia lamblia is present in the intestinal lumen as a binucleate, flagellated trophozoite or a quadranucleate, immotile cyst. Here we used the plant lectin wheat germ agglutinin (WGA), which binds to the disaccharide di-N-acetyl-chitobiose (GlcNAc₂), which is the truncated Asn-linked glycan (N-glycan) of Giardia, to affinity purify the N-glycomes (glycoproteins with N-glycans) of trophozoites and cysts. Fluorescent WGA bound to the perinuclear membranes, peripheral acidified vesicles, and plasma membranes of trophozoites. In contrast, WGA bound strongly to membranes adjacent to the wall of Giardia cysts and less strongly to the endoplasmic reticulum and acidified vesicles. WGA lectin-affinity chromatography dramatically enriched secreted and membrane proteins of Giardia, including proteases and acid phosphatases that retain their activities. With mass spectroscopy, we identified 91 glycopeptides with N-glycans and 194 trophozoite-secreted and membrane proteins, including 42 unique proteins. The Giardia oligosaccharyltransferase, which contains a single catalytic subunit, preferred N glycosylation sites with Thr to those with Ser in vivo but had no preference for flanking amino acids. The most-abundant glycoproteins in the N-glycome of trophozoites were lysosomal enzymes, folding-associated proteins, and unique transmembrane proteins with Cys-, Leu-, or Gly-rich repeats. We identified 157 secreted and membrane proteins in the Giardia cysts, including 20 unique proteins. Compared to trophozoites, cysts were enriched in Gly-rich repeat transmembrane proteins, cyst wall proteins, and unique membrane proteins but had relatively fewer Leu-rich repeat proteins, folding-associated proteins, and unique secreted proteins. In summary, there are major changes in the Giardia N-glycome with the differentiation from trophozoites to cysts.     

Carbohydrate analysis of Acanthamoeba castellanii

We analyzed biochemically Acanthamoeba castellanii trophozoites, intact cysts and cyst walls belonging to the T4 genotype using gas chromatography combined with mass spectrometry. Cyst walls were prepared by removing intracellular material from cysts by pre-treating them with sodium dodecyl sulphate (SDS) containing dithiothreitol, and then subjecting these to a series of sequential enzymatic digestions using amyloglucosidase, papain, DNase, RNase and proteinase K. The resulting “cyst wall” material was subsequently lyophilized and subjected to glycosyl composition analysis. Transmission electron microscopy confirmed the removal of intracystic material following enzymatic treatment. Our results showed that treated A. castellanii trophozoites, intact cysts and cyst walls contained various sugar moieties, of which a high percentage was galactose and glucose, in addition to small amounts of mannose, and xylose. Linkage analysis revealed several types of glycosidic linkages including the 1,4-linked glucosyl conformation, indicative of cellulose. Inhibitor studies suggested that, beside sugar synthesis, cytoskeletal re-arrangement and mitogen-activated protein kinase-mediated pathways are involved in A. castellanii encystment.     

Comparison of Levels of Inactivation of Two Isolates of Giardia lamblia Cysts by UV Light

The effects of 254-nm UV irradiation on two human isolates (WB and H3) of Giardia lamblia cysts were assessed using a collimated beam protocol and a Mongolian gerbil model. The levels of infection of cysts in the gerbils were assessed based on the presence of cysts in feces and the presence and activity of trophozoites in the small intestine of inoculated gerbils. The results suggest that there were differences in the infectivities of the WB and H3 isolates, as well as in susceptibilities of the parasites to UV light. Without UV exposure, gerbils were more readily infected by isolate H3 cysts. After UV exposure of the cysts, however, the gerbils were more susceptible to isolate WB cysts.     

Identification of Giardia lamblia DHHC Proteins and the Role of Protein S-palmitoylation in the Encystation Process

Protein S-palmitoylation, a hydrophobic post-translational modification, is performed by protein acyltransferases that have a common DHHC Cys-rich domain (DHHC proteins), and provides a regulatory switch for protein membrane association. In this work, we analyzed the presence of DHHC proteins in the protozoa parasite Giardia lamblia and the function of the reversible S-palmitoylation of proteins during parasite differentiation into cyst. Two specific events were observed: encysting cells displayed a larger amount of palmitoylated proteins, and parasites treated with palmitoylation inhibitors produced a reduced number of mature cysts. With bioinformatics tools, we found nine DHHC proteins, potential protein acyltransferases, in the Giardia proteome. These proteins displayed a conserved structure when compared to different organisms and are distributed in different monophyletic clades. Although all Giardia DHHC proteins were found to be present in trophozoites and encysting cells, these proteins showed a different intracellular localization in trophozoites and seemed to be differently involved in the encystation process when they were overexpressed. dhhc transgenic parasites showed a different pattern of cyst wall protein expression and yielded different amounts of mature cysts when they were induced to encyst. Our findings disclosed some important issues regarding the role of DHHC proteins and palmitoylation during Giardia encystation.     

The use of irradiated vaccine in immunization against experimental murine toxoplasmosis.

Trophozoites from the peritoneal cavities of mice infected with the RH strain Toxoplasma gondii were given irradiation in doses of 5, 10, 15, and 20 kiloroentgens (kr). CD-1 strain mice that received intraperitoneal inoculation of trophozoites irradiated with 5 kr all died of toxoplasmosis, but the mice that received trophozoites irradiated with the higher doses all survived. The survirors that were examined were found to be free of toxoplasmic cysts. Single doses of these irradiated vaccines provided good protection to subsequent virulent challenge. This protection was 100% in the first 3 weeks after the immunization. Survivors of the first challenge were also solidly protected against a subsequent rechallenge.