Purification of Giardia muris cysts by velocity sedimentation

Giardia muris cysts were separated from fecal contaminants in primary isolates by unit gravity velocity sedimentation. Crude isolates obtained by centrifugation over 1.0 M sucrose were overlaid onto a Percoll density gradient, 1.01 to 1.03 g/ml. G. muris cysts were well separated from faster-sedimenting fecal debris and slower-sedimenting Spironucleus muris and bacteria in 1.5 h.     

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.     

The detection of Giardia muris and Giardia lamblia cysts by immunofluorescence in animal tissues and fecal samples subjected to cycles of freezing and thawing

The effects of freezing and thawing on the detection of selected Giardia spp. cysts were investigated using immunofluorescence, bright field microscopy, and low voltage scanning electron microscopy (SEM). Giardia muris cysts were obtained from either animal carcasses, fecal pellets, or isolated cyst preparations, whereas Giardia lamblia cysts were isolated from fecal samples. These samples were stained using an immunofluorescence technique after 1-3 freezing (-16 C) and thawing (20 C) cycles. Cysts were detected successfully by immunofluorescence in all samples. However, in those samples subjected to freeze-thawing, the cyst walls often became distorted and then were not detectable by bright field microscopy. Low voltage SEM demonstrated that the filaments in the distorted cyst wall underwent rearrangements of interfilament spacing. Quantitation of cyst recovery after freezing and thawing demonstrated that a substantial loss occurred after 1 cycle of alternating temperature when low concentrations of cysts were used, but not with high concentrations of cysts. Cyst recovery, after 3 freezing and thawing cycles, was dramatically lowered irrespective of the initial cyst concentration. These results demonstrated that immunofluorescence was an effective technique for the detection of Giardia spp. cysts in frozen samples and would suggest that freezing and thawing of fecal samples could prevent the detection of cysts when only bright field microscopy was employed.     

Axenic isolation of viable Giardia muris trophozoites

Large numbers of viable Giardia muris trophozoites were isolated from the duodenum of experimentally infected mice 6 days after inoculation with 1,000 G. muris cysts. A series of shaking, incubation, and washing steps in the presence of the broad-spectrum antibiotic piperacillin readily provided 4.9 +/- 1.5 x 10(5) G. muris trophozoites per mouse, free of detectable contaminant organisms. Anaerobic and microaerophilic culturing and scanning electron microscopy demonstrated axenic status and high purity of the isolates. The viability of trophozoites was 98 +/- 2%. Application of this technique should permit novel immunological and epidemiological analyses of G. muris infection and biochemical investigations of this protozoan parasite.     

Genotyping of Giardia lamblia isolates from humans in China and Korea using ribosomal DNA Sequences

Genetic characterization of a total of 15 Giardia lamblia isolates, 8 from Anhui Province, China (all from purified cysts) and 7 from Seoul, Korea (2 from axenic cultures and 5 from purified cysts), was performed by polymerase chain reaction amplification and sequencing of a 295-bp region near the 5' end of the small subunit ribosomal DNA (eukaryotic 16S rDNA). Phylogenetic analyses were subsequently conducted using sequence data obtained in this study, as well as sequences published from other Giardia isolates. The maximum parsimony method revealed that G. lamblia isolates from humans in China and Korea are divided into 2 major lineages, assemblages A and B. All 7 Korean isolates were grouped into assemblage A, whereas 4 Chinese isolates were grouped into assemblage A and 4 into assemblage B. Two Giardia microti isolates and 2 dog-derived Giardia isolates also grouped into assemblage B, whereas Giardia ardeae and Giardia muris were unique.     

In Vitro Excystation of Spironucleus muris

ABSTRACT. In vitro excystation of Spironucleus muris cysts, purified by sequential sucrose and Percoll gradients from mouse feces, was studied. Three in vitro excystation procedures, used for Giardia , were assessed to determine the most useful method. Excystation was monitored by light microscopy and subsequently characterized by transmission and scanning electron microscopy. Spironucleus muris excysted routinely at a level greater than 90% when induced in Hanks' balanced salt solution containing sodium bicarbonate at pH 2.0 and transferred to Tyrodes' salt solution as an excystation medium. Similarly, high rates of excystation were recorded after induction of S. muris cysts in 0.1 M potassium phosphate buffer (pH 7.0) with sodium bicarbonate and excystation in trypticase-yeast extract-iron medium (TYI medium) or phosphate-buffered saline. A lower rate and percentage of excystation were observed after induction of S. muris cysts in an aqueous hydrochloric acid solution (pH 2.0) followed by excystation in TYI medium. All excystation methods produced extremely active S. muris trophozoites with normal morphology. Nonexcysting S. muris cysts have a wall composed of an outer fibrous and an inner membranous portion. Following induction, numerous vesicles appeared in the peritrophic space. Excystation began by the cyst wall opening at one pole, and the anterior part of the trophozoite protruding from the cyst wall. The trophozoite emerged progressively from the cyst wall and the empty cyst wall appeared to collapse. Excysted trophozoites exhibited normal morphological features of S. muris trophozoites isolated from the mouse intestine.     

Morphology of the cyst of Giardia microti by light and electron microscopy

Cysts of Giardia microti, isolated from feces and intestinal contents of Microtus ochrogaster, were examined by light and electron microscopy. These cysts differed morphologically from cysts of other G. duodenalis morphological types in that these cysts often contained two apparently differentiated trophozoites with mature ventral discs. Cysts more closely resembling those reported for G. lamblia and G. muris were in greater abundance in preparations made from intestinal contents and were interpreted as immature cysts. "Multiple fission" cysts, reported in G. muris and G. microti by earlier workers, were not observed; however, endosymbiotic bacteria were found in the cysts of G. microti and may have been responsible for reports of multiple fission in the cysts of Giardia.     

Development of species-specific rDNA probes for Giardia by multiple fluorescent in situ hybridization combined with immunocytochemical identification of cyst wall antigens.

In this study, we describe the development of fluorescent oligonucleotide probes to variable regions in the small subunit of 16S rRNA in three distinct Giardia species. Sense and antisense probes (17-22 mer) to variable regions 1, 3, and 8 were labeled with digoxygenin or selected fluorochomes (FluorX, Cy3, or Cy5). Optimal results were obtained with fluorochome-labeled oligonucleotides for detection of rRNA in Giardia cysts. Specificity of fluorescent in situ hybridization (FISH) was shown using RNase digestion and high stringency to diminish the hybridization signal, and oligonucleotide probes for rRNA in Giardia lamblia, Giardia muris, and Giardia ardeae were shown to specifically stain rRNA only within cysts or trophozoites of those species. The fluorescent oligonucleotide specific for rRNA in human isolates of Giardia was positive for ten different strains. A method for simultaneous FISH detection of cysts using fluorescent antibody (genotype marker) and two oligonucleotide probes (species marker) permitted visualization of G. lamblia and G. muris cysts in the same preparation. Testing of an environmental water sample revealed the presence of FISH-positive G. lamblia cysts with a specific rDNA probe for rRNA, while negative cysts were presumed to be of animal or bird origin.     

Identification of novel Cryptosporidium genotypes from avian hosts

A total of 430 avian-derived fecal specimens were randomly collected from selected Western Australian commercial aviaries, poultry farms, hatcheries, wildlife parks, and the Perth Zoo and screened for the presence of Cryptosporidium by PCR. Of these, 27 Cryptosporidium-positive isolates were detected, characterized, and compared with 11 avian-derived isolates from the Czech Republic at the 18S rRNA and actin gene loci. Sequence and phylogenetic analysis identified four genetically distinct genotypes, avian genotypes I to IV, from various avian hosts. In addition, the host range for Cryptosporidium galli was extended. Cryptosporidium muris and Cryptosporidium andersoni were also identified in a tawny frogmouth and a quail-crested wood partridge, respectively.     

A new method to determine Giardia cyst viability: correlation of fluorescein diacetate and propidium iodide staining with animal infectivity

The viability of Giardia muris cysts was studied with the fluorogenic dyes fluorescein diacetate (FDA) and propidium iodide (PI). G. muris cysts were seen to fluoresce intensely green with FDA at an excitation wavelength of 450 to 490 nm. Cysts stained with PI fluoresced bright orange at an excitation wavelength of 450 to 490 nm and bright red at 545 to 546 nm. Examination of isolated G. muris cyst preparations stained with FDA-PI revealed that greater than 85% of the cysts stained green with FDA and less than 15% stained orange-red with PI. Using the mouse model for giardiasis, we inoculated FDA- or PI-stained cysts into neonatal mice. Feces were examined at days 3, 5, 8, and 11 postinoculation for the presence of cysts. Using 1,000 FDA-stained cysts as the inoculum, we detected cysts at days 5, 8, and 11 postinoculation in 19 of 19 mice, whereas a 50-fold greater dose of cysts produced infection in 27 of 27 mice at day 3 as well as at days 5, 8, and 11 postinoculation. Inoculation of mice with either 5,000 or 50,000 PI-stained G. muris cysts did not produce infection in any of the animals. Necropsy of mice infected with FDA-stained cysts showed trophozoites within the intestines. No trophozoites were detected within animals inoculated with PI-stained cysts. These results demonstrate that FDA-positive cysts are viable, as determined by infectivity, while PI-positive cysts are nonviable and incapable of producing G. muris infections in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new method to determine Giardia cyst viability: correlation of fluorescein diacetate and propidium iodide staining with animal infectivity.

The viability of Giardia muris cysts was studied with the fluorogenic dyes fluorescein diacetate (FDA) and propidium iodide (PI). G. muris cysts were seen to fluoresce intensely green with FDA at an excitation wavelength of 450 to 490 nm. Cysts stained with PI fluoresced bright orange at an excitation wavelength of 450 to 490 nm and bright red at 545 to 546 nm. Examination of isolated G. muris cyst preparations stained with FDA-PI revealed that greater than 85% of the cysts stained green with FDA and less than 15% stained orange-red with PI. Using the mouse model for giardiasis, we inoculated FDA- or PI-stained cysts into neonatal mice. Feces were examined at days 3, 5, 8, and 11 postinoculation for the presence of cysts. Using 1,000 FDA-stained cysts as the inoculum, we detected cysts at days 5, 8, and 11 postinoculation in 19 of 19 mice, whereas a 50-fold greater dose of cysts produced infection in 27 of 27 mice at day 3 as well as at days 5, 8, and 11 postinoculation. Inoculation of mice with either 5,000 or 50,000 PI-stained G. muris cysts did not produce infection in any of the animals. Necropsy of mice infected with FDA-stained cysts showed trophozoites within the intestines. No trophozoites were detected within animals inoculated with PI-stained cysts. These results demonstrate that FDA-positive cysts are viable, as determined by infectivity, while PI-positive cysts are nonviable and incapable of producing G. muris infections in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of Giardia microti and Spironucleus muris cysts in the vole: an immunocytochemical, light, and electron microscopic study

We have shown that cysts of the genus Spironucleus share many common morphological features with Giardia cysts including: 2-4 nuclei, flagellar axonemes, a distinct cyst wall, and they even display the same immunostaining as Giardia cysts when labeled with antibodies specific for Giardia cyst wall. A direct comparison of Spironucleus muris and Giardia microti cysts have revealed that cysts of S. muris are significantly smaller than cysts of G. miroti. At the ultrastructural level, the cyst walls are similar in fibrillar appearance, but the width of the S. muris cyst wall is significantly less than that of G. microti. The cysts of S. muris also differ from G. microti in that they contain a striated rootlet fiber, flagellar sheath, and numerous glycogen rosettes. Characteristic features of Giardia include the adhesive disc and median body. Although the cysts of Spironucleus and Giardia are similar in appearance, these unique morphological features can be used to distinguish between the 2 protozoa and should be employed in the detection of Giardia cysts in water samples.     

Viability of Giardia cysts suspended in lake, river, and tap water.

Numerous waterborne outbreaks of giardiasis have occurred since 1965, yet little or no information has been reported on the viability of Giardia cysts in different aquatic environments. We have studied the viability of Giardia muris cysts suspended in lake, river, and tap water, while also monitoring water temperature, dissolved oxygen, pH, and other water quality parameters. Fecal pellets containing G. muris cysts were placed in glass vials covered with filter paper and exposed to (i) lake water at 15 ft (ca. 4.6 m) and 30 ft (ca. 9.2 m), (ii) river water, (iii) tap water, and (iv) distilled water stored under laboratory conditions. At 3, 7, 14, 28, 56, and 84 days, two vials from each environment were removed, and cyst viability was determined by (i) fluorogenic dye exclusion, (ii) production of giardiasis in an animal, and (iii) cyst morphology by Nomarski microscopy. In the fall, the cysts suspended at 30 ft in lake water remained viable for up to 56 days whereas cysts stored at 15 ft were nonviable after day 28. The G. muris cysts exposed to river water remained viable up to 28 days as determined by the production of giardiasis in mice. G. muris cysts suspended in tap water showed no signs of viability after 14 days, while cysts serving as controls (exposed to refrigerated distilled water) remained viable for up to 56 days. In the winter, Giardia cysts suspended in either lake or river water were viable for 56 to 84 days whereas cysts exposed to tap water were nonviable by day 14.(ABSTRACT TRUNCATED AT 250 WORDS)     

Viability of Giardia cysts suspended in lake, river, and tap water

Numerous waterborne outbreaks of giardiasis have occurred since 1965, yet little or no information has been reported on the viability of Giardia cysts in different aquatic environments. We have studied the viability of Giardia muris cysts suspended in lake, river, and tap water, while also monitoring water temperature, dissolved oxygen, pH, and other water quality parameters. Fecal pellets containing G. muris cysts were placed in glass vials covered with filter paper and exposed to (i) lake water at 15 ft (ca. 4.6 m) and 30 ft (ca. 9.2 m), (ii) river water, (iii) tap water, and (iv) distilled water stored under laboratory conditions. At 3, 7, 14, 28, 56, and 84 days, two vials from each environment were removed, and cyst viability was determined by (i) fluorogenic dye exclusion, (ii) production of giardiasis in an animal, and (iii) cyst morphology by Nomarski microscopy. In the fall, the cysts suspended at 30 ft in lake water remained viable for up to 56 days whereas cysts stored at 15 ft were nonviable after day 28. The G. muris cysts exposed to river water remained viable up to 28 days as determined by the production of giardiasis in mice. G. muris cysts suspended in tap water showed no signs of viability after 14 days, while cysts serving as controls (exposed to refrigerated distilled water) remained viable for up to 56 days. In the winter, Giardia cysts suspended in either lake or river water were viable for 56 to 84 days whereas cysts exposed to tap water were nonviable by day 14.(ABSTRACT TRUNCATED AT 250 WORDS)     

The ferret (Putorius putorius furo), an additional final host of Sarcocystis muris (author's transl)]

Two ferrets were fed mice experimentally infected with Sarcocystis muris. After 7 days they excreted with their faeces for 9 days sporocysts which were morphologically indistinguishable from S. muris sporocysts. Five mice which each received 70 of these sporocysts orally developed macroscopically visible cysts of S. muris in their musculature after 4 months.     

Morphological and biochemical correlates in the characterization of Sarcocystis spp

Isoenzyme electrophoretic techniques were applied to the characterization of seven Sarcocystis spp. that had been identified by conventional morphological studies. Cystozoites were harvested from macroscopic cysts from sheep, cattle, and mice and from microscopic cysts from sheep, cattle, and goats. Soluble cystozoite extracts were subjected to cellulose acetate gel electrophoresis and characterized at 15 of the 39 enzyme loci examined. Genetic relationships among isolates were examined by simple phenetic clustering. Two different morphological types of macroscopic cysts from sheep, identified as S. gigantea (syn. S. ovifelis) and S. medusiformis, consistently differed at 40% of the loci examined. Such genetic divergence confirms their separate morphotypic classification. Both differed from microscopic cyst isolates from sheep at 87% of the loci examined; however, two different morphotypes of microscopic cysts were found in the sheep sampled (thick-walled and thin-walled cysts). Until sufficient numbers of each type can be isolated and examined separately, both were regarded as belonging to the species S. tenella (syn. S. ovicanis). Macroscopic and microscopic cysts from cattle consistently differed at 80% of the loci thereby supporting their separate classification as S. hirsuta (syn. S. bovifelis) and S. cruzi (syn. S. bovicanis), respectively. Isolates from goats (microscopic cysts identified as S. capracanis) differed from S. tenella and S. cruzi at 20% and 47% of the loci, respectively. All macroscopic cyst isolates from the various host animal species (including S. muris from mice) differed from each other at nearly all loci. Isoenzyme electrophoretic techniques therefore provided genetic evidence supporting the classification of these various Sarcocystis spp. by their morphological characteristics.     

Propidium iodide as an indicator of Giardia cyst viability.

The use of propidium iodide, whose uptake indicates cell death or damage, was investigated to assess the viability of heat-inactivated and chemically inactivated Giardia muris cysts. This was done by comparing propidium iodide staining with excystation. We first determined that propidium iodide could be used with an immunofluorescence detection procedure by showing that the percentages of Giardia lamblia cysts stained with this dye before and after subjecting them to a fluorescence detection method were similar. G. muris cysts were then exposed to heat (56 degrees C), 0.5 to 4 mg of chlorine per liter (pH 7.0, 5 degrees C), 0.1 to 10 mg of a quaternary ammonium compound per liter, or 2 mg of preformed and forming monochloramine per liter (pH 7.2, 18 to 20 degrees C). A good positive correlation between percent propidium iodide-stained cysts and lack of excystation was demonstrated for G. muris cysts exposed either to heat or to the quaternary ammonium compound. However, no significant correlation between absence of excystation and propidium iodide staining was found for cysts exposed to chlorine or monochloramines. These results demonstrate that the propidium iodide staining procedure is not satisfactory for determining the viability of G. muris cysts exposed to these two commonly used drinking water disinfectants.     

Development of the cyst-forming coccidian Sarcocystis muris in the body of its intermediate host

On examining the cyst merogony of S. muris, two patterns of development were established. The one occurs in young cysts where metrocytes underwent endopolygeny, giving rise to the intermediate cells. No dividing metrocytes were seen in the older cysts. Unlike, metrocyte differentiation was observed in these from 1, 2 and 3 step metrocytes to the intermediate cell state, which is the other pattern of development. An original scheme of development within the cysts of S. muris is proposed.     

Propidium iodide as an indicator of Giardia cyst viability.

The use of propidium iodide, whose uptake indicates cell death or damage, was investigated to assess the viability of heat-inactivated and chemically inactivated Giardia muris cysts. This was done by comparing propidium iodide staining with excystation. We first determined that propidium iodide could be used with an immunofluorescence detection procedure by showing that the percentages of Giardia lamblia cysts stained with this dye before and after subjecting them to a fluorescence detection method were similar. G. muris cysts were then exposed to heat (56 degrees C), 0.5 to 4 mg of chlorine per liter (pH 7.0, 5 degrees C), 0.1 to 10 mg of a quaternary ammonium compound per liter, or 2 mg of preformed and forming monochloramine per liter (pH 7.2, 18 to 20 degrees C). A good positive correlation between percent propidium iodide-stained cysts and lack of excystation was demonstrated for G. muris cysts exposed either to heat or to the quaternary ammonium compound. However, no significant correlation between absence of excystation and propidium iodide staining was found for cysts exposed to chlorine or monochloramines. These results demonstrate that the propidium iodide staining procedure is not satisfactory for determining the viability of G. muris cysts exposed to these two commonly used drinking water disinfectants.     

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)