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Experimental human infections with Giardia lamblia.

Fifteen healthy volunteers were inoculated enterally with trophozoites of two distinct human isolates of Giardia lamblia, GS/M and Isr. Each of two groups of five volunteers were inoculated with 50,000 (GS/M or Isr) trophozoites. All of the volunteers inoculated with GS/M and none of the volunteers inoculated with Isr became infected. Three of five volunteers infected with GS/M became ill, including two who had diarrhea and typical symptoms of giardiasis. In the second study, three patients who were previously infected with GS/M and treated were rechallenged 12 weeks after the first inoculation, together with five new control volunteers. All of the latter group became infected, and two developed loose stools; two rechallenged volunteers became reinfected but were asymptomatic, and a third was retrospectively found to be infected at the time of challenge. Serum IgM, IgG, and IgA antibody responses to Giardia and intestinal fluid IgA antibody responses to Giardia occurred in 100%, 70%, 60%, and 50%, respectively, of infected volunteers. These studies fulfill Koch's postulates and demonstrate strain variation in the pathogenicity of Giardia infections in humans.

Adult↗

Enterococcus faecium SF68 enhances the immune response to Giardia intestinalis in mice.

We studied the ability of the probiotic organism Enterococcus faecium SF68 to antagonize Giardia intestinalis infection in mice. Oral feeding of E. faecium strain SF68 starting 7 d before inoculation with Giardia trophozoites significantly increased the production of specific anti-Giardia intestinal IgA and blood IgG. This humoral response was mirrored at the cellular level by an increased percentage of CD4(+) T cells in the Peyer's patches and in the spleens of SF68-fed mice. The improvement of specific immune responses in probiotic-fed mice was associated with a diminution in the number of active trophozoites in the small intestine as well as decreased shedding of fecal Giardia antigens (GSA65 protein). The ability of SF68 to stimulate the immune system at both mucosal and systemic levels highlights mechanisms by which this probiotic might antagonize pathogens in vivo. Taken together, the data demonstrate the strong potential of strain SF68 to prevent protozoa from causing intestinal infections.

Animals↗

The Giardia lamblia trophozoite contains sets of closely related chromosomes.

Size variations in homologous chromosomes from six Giardia lamblia isolates have been demonstrated. Four or five intensely stained (major) bands as well as a variable number of lightly stained (minor) bands are present in pulsed field gradient separations. Southern blot analysis with total chromosomal DNA as well as chromosome specific probes indicates that each minor band cross-hybridizes with a major band. Minor bands of doubly cloned organisms appear identical to those of parent clones, indicating that the minor bands do not reflect the presence of variant members within the total population of trophozoites. Densitometric comparisons of chromosome bands from known numbers of Plasmodium falciparum ring stage forms and known numbers of Giardia trophozoites suggest that minor bands MBa and MBb are present in each Giardia trophozoite. Comparison of Not I restriction fragments from the major and minor bands reveals common restriction fragments. Taken together, the data imply that sets of closely related chromosomes occur in the Giardia trophozoite.

Animals↗

Intestinal inflammation measured by fecal neopterin in Gambian children with enteropathy: association with growth failure, Giardia lamblia, and intestinal permeability.

OBJECTIVES: Investigate whether fecal neopterin concentration (a potential marker of gut inflammation) in Gambian children with enteropathy was associated with growth failure. Secondary outcome measures tested the associations between Giardia lamblia infestation, fecal neopterin and lactulose mannitol absorption ratio(L:M), a measure of intestinal permeability. METHODS: Seventy-two children had height and weight measured every 6 to 8 weeks until 15 months of age in a rural Gambian village. L:M ratio, a measure of small intestinal permeability and fecal neopterin were measured at these times. Stool was examined by immunofluorescence and light microscope for Giardia cysts. RESULTS: Long-term height and weight gains were negatively associated with mean subject fecal neopterin concentration (r = -0.29 and -0.36, respectively; P < 0.001). There was no correlation between fecal neopterin and intestinal permeability or history of diarrhea. Of 72 children studied, 19 had Giardia cysts in stool and 38 had negative stool examinations. Infected children had a mean of 0.7 days of diarrhea/week (95% confidence interval [CI], 0.31-1.03) versus 0.8 days/week (95% CI, 0.71-0.85) in uninfected children. No difference in growth was detected between those with positive or negative fecal smears. Mean L:M ratio was the same in both groups (0.31; 95% CI, 0.26-0.34). CONCLUSIONS: Consistent with the theory that intestinal inflammation in tropical infants may impair growth, fecal neopterin concentrations were inversely associated with growth. Factors other than Giardia are causing enteropathy and growth failure in Gambian infants.

Animals↗

Cyst wall synthase: N-acetylgalactosaminyltransferase activity is induced to form the novel N-acetylgalactosamine polysaccharide in the Giardia cyst wall.

Uridine-5'-diphospho-N-acetylgalactosamine (UDP-GalNAc) is required in the formation of the outer filamentous wall of Giardia and is synthesized by inducible enzymes in the cytosol of encysting trophozoites. In this study, an inducible enzyme activity that is associated with a particle population isolated from encysting Giardia is reported, and this activity exclusively incorporates [1-(14)C]GalNAc (from UDP-[(14)C]GalNAc) into an ethanol precipitate with the same properties as the filamentous cyst wall of GIARDIA: This ethanol precipitate exhibits characteristics of Giardia cyst wall filaments in that both contain GalNAc as the only sugar moieties and are SDS-insoluble, proteinase- and alkali-resistant and acid-hydrolysable. However, since the precise chemical nature of the ethanol precipitate remains unknown, this enzyme activity is referred to tentatively as cyst wall synthase (CWS). CWS activity peaks in cells between 24 and 36 h of encystment and exhibits a high affinity and marked specificity for UDP-GalNAc as its substrate. UDP-N-acetylglucosamine, UDP-glucose, UDP-galactose, D-glucosamine and D-galactosamine were not incorporated into the ethanol precipitate. Partially purified CWS activity exhibits an apparent K(m) of 0.048 mM for UDP-GalNAc, a V(max) of 0.70 nmol x min(-1) (mg protein)(-1) and a requirement for divalent cations in the following order of preference: Ca(2+), Mg(2+)>Co(2+)>>>Mn(2+), Zn(2+). EDTA inhibits CWS activity.

Acetylgalactosamine↗

Batch solar disinfection inactivates oocysts of Cryptosporidium parvum and cysts of Giardia muris in drinking water.

AIM: To determine whether batch solar disinfection (SODIS) can be used to inactivate oocysts of Cryptosporidium parvum and cysts of Giardia muris in experimentally contaminated water. METHODS AND RESULTS: Suspensions of oocysts and cysts were exposed to simulated global solar irradiation of 830 W m(-2) for different exposure times at a constant temperature of 40 degrees C. Infectivity tests were carried out using CD-1 suckling mice in the Cryptosporidium experiments and newly weaned CD-1 mice in the Giardia experiments. Exposure times of > or =10 h (total optical dose c. 30 kJ) rendered C. parvum oocysts noninfective. Giardia muris cysts were rendered completely noninfective within 4 h (total optical dose >12 kJ). Scanning electron microscopy and viability (4',6-diamidino-2-phenylindole/propidium iodide fluorogenic dyes and excystation) studies on oocysts of C. parvum suggest that inactivation is caused by damage to the oocyst wall. CONCLUSIONS: Results show that cysts of G. muris and oocysts of C. parvum are rendered completely noninfective after batch SODIS exposures of 4 and 10 h (respectively) and is also likely to be effective against waterborne cysts of Giardia lamblia. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate that SODIS is an appropriate household water treatment technology for use as an emergency intervention in aftermath of natural or man-made disasters against not only bacterial but also protozoan pathogens.

Animals↗

A novel palmitoyl acyl transferase controls surface protein palmitoylation and cytotoxicity in Giardia lamblia.

The intestinal protozoan parasite Giardia lamblia undergoes surface antigenic variation whereby one of a family of structurally related variant-specific surface proteins (VSPs) is replaced in a regulated process by another antigenically distinct VSP. All VSPs are type I membrane proteins that have a conserved hydrophobic sequence terminated by the invariant hydrophilic amino acids, CRGKA. Using transfected Giardia constitutively expressing HA-tagged VSPH7 and incubated with radioactive [3H]palmitate, we demonstrate that the palmitate is attached to the Cys in the conserved CRGKA tail. Surface location of mutant VSPs lacking either the CRGKA tail or its Cys is identical to that of wild-type VSPH7 but non-palmitoylated mutants fail to undergo complement-independent antibody specific cytotoxicity. In addition, membrane localization of non-palmitoylated mutant VSPH7 changes from a pattern similar to rafts to non-rafts. Palmitoyl transferases (PAT), responsible for protein palmitoylation in other organisms, often possess a cysteine-rich domain containing a conserved DHHC motif (DHHC-CRD). An open reading frame corresponding to a putative 50 kDa Giardia PAT (gPAT) containing a DHHC-CRD motif was found in the Giardia genome database. Expression of epitope-tagged gPAT using a tetracycline inducible vector localized gPAT to the plasma membrane, a pattern similar to that of VSPs. Transfection with gPAT antisense producing vectors inhibits gPAT expression and palmitoylation of VSPs in vitro confirming the function of gPAT. These results show that VSPs are palmitoylated at the cysteine within the conserved tail by gPAT and indicate an essential function of palmitoylation in control of VSP-mediated signalling and processing.

Acyltransferases↗

Biliary antibody response in rats infected with rodent Giardia duodenalis isolates.

Using a sensitive ELISA, specific serum and bile anti-Giardia IgM and IgA responses were studied in rats infected with two strains of Giardia duodenalis: a rat isolate which produces a chronic infection and a mouse isolate which produces a self-limiting infection. Paired samples of serum and bile were collected from groups of DA (RT1avl) rats at various times during primary and secondary infections. Antibody responses to both organisms were similar. Only IgA anti-Giardia antibodies were detected in bile whereas both IgM and IgA antibodies were detected in serum. Biliary IgA antibody titres increased throughout the course of the primary infection and remained at high levels for at least 10 weeks. Biliary IgA titres increased 16-fold during the secondary infection with both isolates. Serum IgA anti-Giardia titres also increased but more slowly than the titres in bile. Serum IgM antibody responses were observed against both organisms during the primary and secondary infections. Trophozoites harvested from the intestinal lumen during primary infections were examined for surface-bound IgA by immunofluorescence microscopy. IgA was detected on 3% of trophozoites on day 7 after infection but on over 70% of trophozoites by the 10th day. The data demonstrate the occurrence of a secretory IgA immune response in rats infected with both G. duodenalis isolates, some of which is directed against surface antigens of the trophozoites.

Animals↗

A H2O-producing NADH oxidase from the protozoan parasite Giardia duodenalis.

We describe the purification of a H2O-producing NADH oxidase from the protozoan parasite Giardia duodenalis. The enzyme is a monomeric flavoprotein containing flavin adenine dinucleotide in a 1:1 molar ratio with the polypeptide. The NADH oxidase has an apparent molecular mass of 46 kDa and was homogenous as determined by denaturing gel electrophoresis and N-terminal amino acid sequencing. NADPH could substitute for NADH as an electron donor with a K(m) value of 4.2 microM for NADH and 16 microM for NADPH (pH 7.8 at room temperature). With oxygen as the primary electron acceptor under aerobic conditions, the pure enzyme did not produce O.-2 nor H2O2 as stoichiometric products of oxygen reduction, implicating H2O as the end product and obviating the need for superoxide dismutase. The ability to utilise oxygen explains the apparent respiration of the amitochondrial fermentative metabolism of Giardia. Mercurials, flavoantagonists and heavy metals (Cu2+ and Zn2+) inhibited this activity. Under anaerobic conditions the enzyme catalysed electron transfer at lower efficiencies to other electron acceptors including nitroblue tetrazolium, potassium ferricyanide, FAD and FMN, using either NADH or NADPH as electron donors. NADPH, however, was a more efficient electron donor. Cytochrome c was not reduced under any assay conditions used. The enzyme reduced the nitrofuran drugs, furazolidone (an antigiardial) and nitrofurantoin, to their toxic radical forms as determined by EPR. Metronidazole, a nitroimidazole, was not reduced. Pure NADH oxidase did not demonstrate ferredoxin:NAD(P)1 oxidoreductase activity since it could not accept electrons from reduced ferredoxin to regenerate NAD(P)H. The G. duodenalis NADH oxidase may, therefore, function as a terminal oxidase, similar to the mitochondrial cytochrome oxidase, and in the maintenance of an optimum intracellular redox ratio. This report of a flavoenzyme from Giardia places Giardia close to the anaerobic bacteria in evolutionary terms.

Amino Acid Sequence↗

Comparative analysis of Cryptosporidium, Giardia and indicator bacteria during sewage sludge hygienization in various composting processes.

AIMS: To evaluate the suitability of Clostridium perfringens, Escherichia coli and enterococci as indicator organisms for Cryptosporidium and Giardia in treated sludge. METHODS AND RESULTS: Occurrence of Cryptosporidium oocysts and Giardia cysts, detected and enumerated by direct immunofluorescence microscopy, were compared with counts of indicator bacteria during six different sewage sludge hygienization processes, including closed reactor and open windrow composting, and sludge sanitation by quicklime or peat addition. No statistical correlation existed between the counts of indicator bacteria, Cl. perfringens, E. coli, and enterococci and occurrence of Cryptosporidium or Giardia. In sludge end-products, Giardia cysts were detected more frequently than Cryptosporidium oocysts. SIGNIFICANCE OF THE STUDY: Direct analysis is the best method to confirm the presence of (oo)cysts in sludge.

Animals↗

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.

Animals↗

Detection of Microsporidia, cryptosporidia and Giardia in swimming pools: a one-year prospective study.

In order to estimate the rate of microsporidia, cryptosporidia and giardia contamination of swimming pools, sequential samples of water were collected during a one-year period in six different swimming pools in Paris, France. Fourty-eight samples were submitted to filtrations. Eluates were examined for microsporidia using polymerase chain reaction (PCR) and for cryptosporidia and giardia using immunofluorescence staining. One of 48 specimens was positive for microsporidia. Using DNA sequence analysis, unknown microsporidia species were identified, which were close to an insect microsporidia Endoreticulatus schubergi. One sample was positive for cryptosporidia and none were positive for giardia. This study shows a low level of swimming pool water contamination by microsporidia, cryptosporidia or giardia, demonstrating the efficacy of cleaning filtration and disinfection procedures used in French swimming pools.

Animals↗

Small rodents and other mammals associated with mountain meadows as reservoirs of Giardia spp. and Campylobacter spp.

Sixty-five percent (469 of 722) of the fecal samples collected from small rodents in the central Washington Cascade mountains were positive for Giardia spp. Trapping studies showed that microtines of the genus Microtus were heavily infected with the parasite. Morphologically the cysts and trophozoites were of the Giardia duodenalis type. Small-rodent populations appear to maintain their infection throughout the year. Our data suggest that there is no difference in the percentage of positive animals in areas receiving a lot of human use as opposed to animals in those areas receiving very little or no human use. Giardia spp. were also found in elk and beaver fecal samples. Campylobacter spp. were recovered infrequently from the small rodents inhabiting alpine meadows. Of 551 specimens cultured, less than 1% were positive for the bacterium, and the isolates were identified as Campylobacter coli. Water voles were susceptible to a human isolate of Campylobacter jejuni and shed the bacterium for several weeks. C. jejuni was also isolated from a bear fecal sample collected from a protected watershed. Our studies indicate that microtines and possibly other small rodents inhabiting mountain meadows have a potential to act as a reservoir for both Giardia spp. and Campylobacter spp. Because these animals may carry human pathogens, they should be included in animal surveys designed to assess the health risks associated with mountain watersheds.

Animals↗

Comparison of two methods for detection of Giardia cysts and Cryptosporidium oocysts in water.

The steps of two immunofluorescent-antibody-based detection methods were evaluated for their efficiencies in detecting Giardia cysts and Cryptosporidium oocysts. The two methods evaluated were the American Society for Testing and Materials proposed test method for Giardia cysts and Cryptosporidium oocysts in low-turbidity water and a procedure employing sampling by membrane filtration, Percoll-Percoll step gradient, and immunofluorescent staining. The membrane filter sampling method was characterized by higher recovery rates in all three types of waters tested: raw surface water, partially treated water from a flocculation basin, and filtered water. Cyst and oocyst recovery efficiencies decreased with increasing water turbidity regardless of the method used. Recoveries of seeded Giardia cysts exceeded those of Cryptosporidium oocysts in all types of water sampled. The sampling step in both methods resulted in the highest loss of seeded cysts and oocysts. Furthermore, much higher recovery efficiencies were obtained when the flotation step was avoided. The membrane filter method, using smaller tubes for flotation, was less time-consuming and cheaper. A serious disadvantage of this method was the lack of confirmation of presumptive cysts and oocysts, leaving the potential for false-positive Giardia and Cryptosporidium counts when cross-reacting algae are present in water samples.

Animals↗

Detection of viable Giardia cysts by amplification of heat shock-induced mRNA.

Primers obtained from gene sequences coding for heat shock proteins (HSP) were used to specifically detect enteric protozoans of the genus Giardia. The HSP primers amplified Giardia DNA or the corresponding RNA sequences obtained from lysed cysts and gave a 163-bp product. Since the presence of the product did not indicate whether the cysts were viable, these amplifications are a presence/absence test only. In contrast, amplification of heat shock-induced mRNA utilizing the same HSP primers was indicative of viable Giardia cysts. The limit of sensitivity of the presence/absence test was 1 cyst, whereas for the viability test it was 10 cysts. Thus, viable Giardia cysts can be rapidly and specifically detected with great sensitivity through the use of PCR amplifications.

Animals↗

Sheep may not be an important zoonotic reservoir for Cryptosporidium and Giardia parasites.

Little is known of the prevalence of Cryptosporidium and Giardia parasites in sheep and the genotypes that they harbor, although potentially sheep may contribute significantly to contamination of watersheds. In the present study, conducted in Western Australia, a total of 1,647 sheep fecal samples were screened for the presence of Cryptosporidium and Giardia spp. using microscopy, and a subset (n = 500) were screened by PCR and genotyped. Analysis revealed that although both parasites were detected in a high proportion of samples by PCR (44% and 26% for Giardia and Cryptosporidium spp., respectively), with the exception of one Cryptosporidium hominis isolate, the majority of isolates genotyped are not commonly found in humans. These results suggest that the public health risk of sheep-derived Cryptosporidium and Giardia spp. in catchment areas and effluent may be overestimated and warrant further investigation.

Animals↗

Biology of Giardia lamblia.

Giardia lamblia is a common cause of diarrhea in humans and other mammals throughout the world. It can be distinguished from other Giardia species by light or electron microscopy. The two major genotypes of G. lamblia that infect humans are so different genetically and biologically that they may warrant separate species or subspecies designations. Trophozoites have nuclei and a well-developed cytoskeleton but lack mitochondria, peroxisomes, and the components of oxidative phosphorylation. They have an endomembrane system with at least some characteristics of the Golgi complex and encoplasmic reticulum, which becomes more extensive in encysting organisms. The primitive nature of the organelles and metabolism, as well as small-subunit rRNA phylogeny, has led to the proposal that Giardia spp. are among the most primitive eukaryotes. G. lamblia probably has a ploidy of 4 and a genome size of approximately 10 to 12 Mb divided among five chromosomes. Most genes have short 5' and 3' untranslated regions and promoter regions that are near the initiation codon. Trophozoites exhibit antigenic variation of an extensive repertoire of cysteine-rich variant-specific surface proteins. Expression is allele specific, and changes in expression from one vsp gene to another have not been associated with sequence alterations or gene rearrangements. The Giardia genome project promises to greatly increase our understanding of this interesting and enigmatic organism.

Animals↗

Cell division of Giardia intestinalis: flagellar developmental cycle involves transformation and exchange of flagella between mastigonts of a diplomonad cell.

Giardia intestinalis is a binucleated diplomonad possessing four pairs of flagella of distinct location and function. Its pathogenic potential depends on the integrity of a complex microtubular cytoskeleton that undergoes a profound but poorly understood reorganization during cell division. We examined the cell division of G. intestinalis with the aid of light and electron microscopy and immunofluorescence methods and present here new observations on the reorganization of the flagellar apparatus in the dividing Giardia. Our results demonstrated the presence of a flagellar maturation process during which the flagella migrate, assume different position, and transform to different flagellar types in progeny until their maturation is completed. For each newly assembled flagellum it takes three cell cycles to become mature. The mature flagellum of Giardia is the caudal one that possesses a privileged basal body at which the microtubules of the adhesive disk nucleate. In contrast to generally accepted assumption that each of the two diplomonad mastigonts develops separately, we found that they are developmentally linked, exchanging their cytoskeletal components at the early phase of mitosis. The presence of the flagellar maturation process in a metamonad protist Giardia suggests that the basal body or centriole maturation is a universal phenomenon that may represent one of the core processes in a eukaryotic cell.

Animals↗