Failure to propagate Cryptosporidium spp. in cell-free culture

The successful propagation of Cryptosporidium parvum in cell-free culture medium was recently reported. To investigate whether this phenomenon could be broadened to include other C. parvum isolates, as well as Cryptosporidium hominis, we attempted to propagate 3 isolates in cell-free medium under reported culture conditions. Cryptosporidium oocysts from C. parvum strains Moredun (MD) or IOWA or C. hominis strain TU502 were added to media containing coagulated newborn calf serum. The cultures were sampled at various times throughout a 45 (IOWA) or 78 (MD, TU502)-day period and were microscopically examined for various life stages of Cryptosporidium. Cell-free cultures harvested on days 45 and 68 postinoculation were tested for in vitro infectivity on Madrin-Darby bovine kidney cells. In vivo infectivity testing was performed using either infant or 2-wk-old immunosuppressed C57BL mice with cell-free cultures harvested on days 52 and 78. Fecal and gut samples collected from mice were examined by modified acid-fast staining. Data from wet mounts, electron microscopy, and in vitro and in vivo infectivity testing showed that the original oocysts did not complete their life cycle and produce new, viable, infectious oocysts in cell-free culture. Thus, we conclude that this is not a universal phenomenon or readily accomplished.     

Protracted shedding of oocysts of Neospora caninum by a naturally infected foxhound

Feces from 15 dogs at 2 different foxhound kennels in the U.K. were examined microscopically for the presence of oocysts of Neospora caninum. One sample containing approximately 400 candidate oocysts per gram was positive in a polymerase chain reaction (PCR) using N. caninum-specific primers. In a sample taken 4 mo later from the same hound. N. caninum oocysts were again detected visually and by PCR. This is the third reported case of a dog naturally excreting oocysts of N. caninum and suggests that oocyst excretion can occur over a relatively long period of time in some circumstances or that reshedding may occur.     

Immunohistochemistry based assay to determine the effects of treatments on Cryptosporidium parvum viability

Cell culture infectivity assays can provide an accurate means of detecting viable Cryptosporidium parvum oocysts from environmental samples or to test the effects of various treatments on oocyst infectivity. Cell culture assays can also be used to test candidate chemotherapeutic agents. The use of a human cell line provides a situation close to human infection. The present assay uses an anti-Cryptospordium primary antibody, combined with a biotinylated secondary antibody, and an immunoperoxidase detection system. Cryptosporidium parvum oocysts excysted in vitro when placed on monolayers of HCT-8 cells and developmental stages including schizonts and merozoites were visualized using light microscopy of the immunoperoxidase stained slides and by transmission electron microscopy of infected HCT-8 cell cultures. Because the immunoperoxidase system used gives a permanent preparation, the cell cultures can be retained and examined later. Dose titration of oocysts indicated that as few as 50 inoculated oocysts could be detected. The activity of paromomycin was evaluated in this system and 500 microg/ml produced a 97.8% reduction in infection.     

Isolation and identification of Cryptosporidium from various animals in Korea. II. Identification of Cryptosporidium muris from mice

Each of SPF mice(Scl: ICR strain, 3-week-old males) was inoculated with 5 x 10(4) oocysts of Cryptosporidium by stomach tube. The oocysts were large type one which was previously isolated from Korean mice, and passaged in 3-week-old SPF mice. The patterns of oocyst discharge were monitored daily, and in order to observe the ultrastructure of developmental stages the stomach of the mice was examined by transmission electron microscopy (TEM) at 4 weeks post-inoculation. The prepatent period for 6 mice was 5.6 days post-inoculation on the average, and the patent period was 63.2 days. The number of oocysts discharged per day from the mice reached peak on day 36.6 post-inoculation on the average. A large number of oocysts were found in fecal samples obtained from inoculated mice on days 30-50 post-inoculation. C. muris was larger than C. parvum at almost every developmental stages, the size difference being 1.4 times in oocysts, 2.4 times in sporozoites, 1.6 times in merozoites, and 1.5 times in microgametes. The ultrastructural features of the attachment site of C. muris to the mucus cells were remarkably different from those of C. parvum and its closely related species. The anterior projection of the protozoa (C. muris), the outer aspect of which was surrounded by a thick filamentous process of the host cell, has not been reported at any developmental stages of C. parvum or its closely related species. The size of the oocysts of strain RN 66 was larger than that of Korean mice origin. The above results reveal that the large type Cryptosporidium of Korean mice origin is identified as Cryptosporidium muris and this type was named as C. muris (strain MCR).     

Characterization of a coelomic gregarine parasite from Thitarodes pui (Lepidoptera: Hepialidae) in the Tibetan Plateau

Thitarodes pui, one of the host species of entomopathogenic fungus Ophiocordyceps sinensis, has great economic importance in the Tibetan Plateau. We report here, for the first time, a gregarine parasite found in the coelom of 7th instar and adults of T. pui. Gregarine gamonts (ovoid, ～15×8μm) underwent syzygy to produce reproductive gametocysts in T. pui larval hemolymph. All infected T. pui carried 2-17 mature gametocysts filled with numerous oocysts (lemon-shaped, 17.17±0.73×6.49±0.4μm). Transmission electron microscopy showed that these oocysts contained vacuoles of various sizes and amylopectin granules in the cytoplasm; scanning electron microscopy revealed a number of small bumps all over the surface of these oocysts. Small subunit ribosomal DNA sequence analysis showed a close relationship between the gregarine and the species of Ascogregarina (Eugregarinorida: Lecudinidae). Internal transcribed spacers and 5.8S ribosomal DNA from this gregarine exhibited 76% highest sequence identity with that from Ascogregarina culicis Ross.     

Synaptonemal complex karyotype of Eimeria tenella

In most organisms, biological variability rests on the behaviour of the chromosomes in the meiotic context. Despite the importance of meiosis, very little is known about the meiotic behaviour of the Eimeria chromosomes. The aim of the present study is to describe the standard synaptonemal complex karyotype from Eimeria tenella oocyst spreads by electron microscopy. For that purpose, complete sets of pachytene synaptonemal complexes were obtained and the morphological pachytene karyotype was determined. The authors used a previously reported method that overcomes the difficulty of the extreme resistance of protozoan oocysts to disruption and permits the release of intact meiotic chromosomes. The chromosomes were selected under a light microscope and those selected were stained with phosphotungtic acid and studied by transmission electron microscopy. The authors confirmed 14 chromosomes, which were observed as synaptonemal complexes, and the karyotype was constructed by arranging synaptonemal complexes according to their relative lengths and kinetochore position. Components of the synaptonemal complex, lateral elements, central element, recombination nodules and kinetochore were observed. Measures of the kynetochore, width of the synaptonemal complex, diameter of the recombination nodule and length of the telomeres are given. Minimal and no significant differences were found between measures of chromosomes isolated from different Eimeria tenella strains. To the best of our knowledge, the present investigation for the first time identifies and describes the morphological characteristics of the synaptonemal complex of Eimeria tenella during the meiosis that occurs within the oocysts. In addition, the authors provide evidence of the presence of recombination nodules, suggesting that the recombination process may play an important role in the molecular evolution of this parasite.     

Improving the rate of infectivity of Cryptosporidium parvum oocysts in cell culture using centrifugation.

Centrifugation was evaluated as a method to improve infectivity assays of Cryptosporidium parvum in cell culture using the focus detection method, an immunofluorescence-based method for detecting infectious C. parvum oocysts in vitro. Human ileocecal adenocarcinoma (HCT-8) cells were grown for 48 hr on 13-mm cover slips in 24-well microtiter plates and infected with bleach-treated C. parvum oocysts. Plates were centrifuged at 228 g for 10 min and incubated at 37 C for 5, 12, 18, 24, and 48 hr. Foci of infection were stained by immunofluorescence and enumerated using epifluorescent microscopy. Results were compared to noncentrifuged controls. Foci in centrifuged samples could be enumerated after 18 hr. According to most probable number (MPN) analysis, the number of infectious oocysts estimated at 48 hr (13,326 infectious oocysts) was reached by 18 hr in centrifuged samples. After 48 hr, there was no significant difference (P < 0.05) between centrifuged and noncentrifuged samples enumerated by number of foci or the MPN of infectious oocysts. Centrifugation may expedite detection during C. parvum infectivity assays. Furthermore, multiwell plate formats are more cost effective than traditional chamber slides.     

Electron Microscopy of Free-Floating Cells: Thin-Layering Technique, and Selection of Individual Cells for Ultramicrotomy

A rapid and accurate procedure for electron microscopy of individual cells from suspensions (blood, peritoneal exudate, etc.) is described. After fixation of the sample with standard techniques, the particulate constituents are suspended in buffered 5% bovine serum albumin, thin-layered by gravity on clear supports (cover glasses or polyester slips) in which an orientation grid had been scored, and then immobilized by exposure of the preparations to acrolein vapors. The specimens are examined for cells of interest under a light microscope using interference or phase contrast; individual cells to be sectioned are documented in three photomicrographs taken at different magnifications. After this the specimens are embedded like ordinary cover slip preparations. When examining the face of the polymerized block under a light microscope, the position of the selected cell beneath the orientation grid relief can readily he relocated by the aid of the pre-embedding reference micrographs.     

Electron microscopy of free-floating cells: thin-layering technique, and selection of individual cells for ultramicrotomy.

A rapid and accurate procedure for electron microscopy of individual cells from suspensions (blood, peritoneal exudate, etc.) is described. After fixation of the sample with standard techniques, the particulate constituents are suspended in buffered 5% bovine serum albumin, thin-layered by gravity on clear supports (cover glasses or polyester slips) in which an orientation grid had been scored, and then immobilized by exposure of the preparations to acrolein vapors. The specimens are examined for cells of interest under a light microscope using interference or phase contrast; individual cells to be sectioned are documented in three photomicrographs taken at different magnifications. After this the specimens are embedded like ordinary cover slip preparations. When examining the face of the polymerized block under a light microscope, the position of the selected cell beneath the orientation grid relief can readily be relocated by the aid of the pre-embedding reference micrographs.     

Comparative study between two laser scanning cytometers and epifluorescence microscopy for the detection of Cryptosporidium oocysts in water.

BACKGROUND: Cryptosporidium detection in water and environmental samples has increased during the last years, largely due to an increase in the number of reported waterborne outbreaks of cryptosporidiosis and the implementation of new regulations about Cryptosporidium monitoring in water supplies. The aim of this study was to validate and compare the capacity of two laser scanning cytometers commercially available (LSC and ChemScanRDI), against manual microscopic enumeration of Cryptosporidium oocysts in surface water and reference material samples. METHODS: Reference material and surface water samples were analysed by two laser scanning cytometers methodologies and by manual epifluorescence microscopy. Two mAbs from commercial suppliers were used to evaluate background reduction. RESULTS: Highly significant correlations were obtain between both cytometers (R(2) = 0.99) and with manual microscopy (R(2) = 0.98), showing that oocysts counts made by cytometers were equivalent to those obtained with conventional methods. We observed a variability in oocysts counts when different antibodies where used with laser scanning cytometers and manual microscopy. CONCLUSIONS: This study showed the efficacy of the laser scanning technology (LSC and ChemScanRDI), as an automated and a more standardized alternative to manual epifluorescence microscopy examination, for Cryptosporidium detection in water samples. High quality antibodies are needed for automated enumeration as well as for manual microscope observations.     

Eimeria indianensis sp. n. and an Isospora sp. from the Opossum Didelphis virginiana (Kerr)*

Two of 15 road-killed opossums examined for coccidia were found to be infected with a hitherto undescribed species of Eimeria, herein named Eimeria indianensis . The oocysts were spherical (63%) or slightly subspherical (37%) with a double-layered wall. The outer layer was ～1.5 μm thick, yellowish, striated, and appeared rough and pitted on the surface. A micropyle was absent. The spherical oocysts were 16.3 (13–18) μm in diameter; the subspherical ones, 17.6 (15–18) × 16.4 (14–17) μm. The sporocysts measured 9.1 (8–10) × 6.2 (6–7) μm and contained a granular residuum. The sporozoites were elongate, measuring 13.4 (13–15) × 1.8 (1.6-2.0) μm; no refractile globules were seen. The prepatent period was 10 days and the patent period ranged from 9–15 days. A few oocysts of an Isospora sp. were present in one opossum. It was not possible to confirm whether they were specifically of the opossum or of spurious origin.     

Sexual and Asexual Development of Cryptosporidium parvum in Five Oocyst– or Sporozoite-Infected Human Enterocytic Cell Lines

ABSTRACT. The human enterocytic cell lines Caco-2, HT29, HCT8 and the Caco-2 clones TC7 and PF11 were studied for their ability to support Cryptosporidium parvum development. Following the addition in cultures of either oocysts or excysted sporozoites, immunofluorescent and transmission electron microscopy revealed the presence of all stages of the parasite life cycle by both procedures, and no difference in the ration of infected cells was found among cell lines. More oocysts were seen in cell monolayers infected with oocysts than with sporozoites (p < 0.0001). The number of meronts observed was the same after either oocysts or sporozoites inoculation. Data suggest that the two methods yield a same cell infection rate.     

Quantification of Cryptosporidium parvum oocysts in mouse fecal specimens using immunomagnetic particles and two-color flow cytometry

Although single-color flow cytometry has been shown to be more sensitive than fluorescence microscopy for the quantification of Cryptosporidium parvum oocysts, this method has not been optimized. Monoclonal antibody OW50, specific to the cell wall of oocysts, was conjugated to superparamagnetic particles, to fluorescein isothiocyanate, and to r-phycoerythrin. The oocysts were then double stained with the fluorochrome-labeled OW50 and were placed in tubes with known numbers of highly fluorescent polystyrene beads, allowing quantification of the oocysts without dependence on acquired sample volume by flow cytometry. Data from 2-color flow cytometry using logical gating of the oocysts and beads showed a linear relationship between dilutions of a purified oocyst suspension and the mean numbers of oocysts detected (r2 = 1.00). An average of 15 purified oocysts/ml were counted in a dilution with a theoretical concentration of 12 oocysts/ml. Known numbers of purified oocysts were seeded into normal mouse fecal specimens, captured by OW50-labeled immunomagnetic particles, eluted with 5% potassium dichromate at low pH, and double stained with fluorochrome-labeled OW50. By flow cytometry, the mean recovery was 43.1% (+/-8.3%), and as few as 133 oocysts were detected. The captured and eluted oocysts were infective in neonatal BALB/c mice. This 2-color flow cytometry method, used in conjunction with the capture and elution of oocysts by and from immunomagnetic particles, provides a powerful tool for not only the quantification and purification of C. parvum oocysts from different sources but also for the characterization of oocysts in vitro and in vivo.     

Neospora caninum-like oocysts observed in feces of free-ranging red foxes (Vulpes vulpes) and coyotes (Canis latrans)

The aim of this study was to examine the feces of free-ranging foxes and coyotes for the presence of Neospora caninum oocysts. Feces were collected from 271 foxes and 185 coyotes in the Canadian province of Prince Edward Island, processed by sucrose flotation, and examined by light microscopy for the presence of coccidian oocysts. In 2 fox and 2 coyote samples, oocysts morphologically and morphometrically similar to oocysts of N. caninum were observed. DNA was extracted from these samples and subjected to nested polymerase chain reaction (PCR) using primers to the N. caninum-specific Nc5 genomic sequence. Through DNA sequencing, alignment of the sequences of at least 3 clones from each isolate to sequences deposited in GenBank revealed 95-99% similarity to the Nc5 sequence of N. caninum. PCR using primers specific for Hammondia heydorni failed to yield an amplification product from these DNA samples.     

Plasmodium yoelii: contribution of oocysts melanization to natural refractoriness in Anopheles dirus

It is well known that Anopheles dirus is naturally refractory to rodent malaria parasite, Plasmodium yoelii, but the mechanism is still largely unknown. Here, we found that some P. yoelii taken into An. dirus could develop into oocysts, but oocysts were partially melanized at 7 days and completely melanized at 15 days post-infectious blood meal. Transmission electronic microscopy could find the melanized P. yoelii oocysts in An. dirus as early as 5 days post-infection, with a few haemocytes attaching to the melanized oocysts, indicating a typical humoral melanization reaction. Although the change of protein pattern at 24h post-infection suggested that other unknown mechanisms and/or factors might be involved in killing ookinetes, our data implied that oocysts melanization was one of the mechanisms of An. dirus to block P. yoelii development. In addition, activity of phenoloxidase, such as monophenol oxidase and o-diphenoloxidase, in haemolymph of An. dirus fed on infectious blood meal was much higher than that of mosquitoes fed on 5% glucose or normal mouse blood (p<0.05), implying the possible role of PO in oocysts melanization by An. dirus.     

An Electron Microscope Study of Autoinfection in Neogregarines (Sporozoa, Neogregarinida)

SYNOPSIS. In an electron microscope study of the neogregarine Farinocystis tribolii Weiser 1953 in the fat body of the larvae of the flour beetle Tribolium castaneum , empty oocysts and adjacent sporozoites of the gregarine were found. The empty oocysts contained host cell cytoplasm, a residuum only slightly larger than that in mature oocysts, and some remnants of the oocyst membrane pressed tightly against the inner surface of the wall. Apparently normal sporozoites were found near the empty oocysts and no damaged ones were seen. It is assumed that the sporozoites go on developing in the host, i.e., that autoinfection takes place.     

Ultrastructure of Excystment of Toxoplasma gondii Oocysts*

SYNOPSIS. The excystation of sporozoites from intact Toxoplasma gondii oocysts or mechanically released sporocysts was studied by light and electron microscopy. Both intact oocysts and free sporocysts excysted in 5% bovine bile in 0.9% NaCl solution after 30–60 min incubation at 37 C. Sporozoites were first activated in either intact sporocysts or oocysts within 2–12 min of incubation in bile. Sporozoites escaped from sporocysts through 4 plate-like sutures in the sporocyst wall, and from the oocyst as the oocyst wall ruptured at one or more points.     

Disseminated toxoplasmosis in a captive ring-tailed lemur (Lemur catta)

A 3-yr-old secundiparous female ring-tailed lemur presented to the Auburn University Small Animal Clinic with signs of dyspnea, lethargy, and anorexia. The animal died before she could be examined, and a full necropsy was immediately performed. Provisional necropsy findings included moderate pneumonia and hepatopathy. Acute interstitial pneumonia and focal hepatocellular necrosis were confirmed histologically. Lung impression smears, histopathology, electron microscopy, immunohistochemistry, and tissue culture isolation resulted in a diagnosis of acute disseminated Toxoplasma gondii infection, which was confirmed by polymerase chain reaction. The isolate of T. gondii was avirulent for mice and was named AU Tgl and genetically is type II. The source of the infection remains unclear, but speculation suggests contaminated fruit or blackbirds (Passeriformes: Icteridae) acting as transport hosts for oocysts from nondomestic felids and feral cats on the property.     

Immunomagnetic separation of Cryptosporidium parvum oocysts using MACS MicroBeads and high gradient separation columns

We evaluated the MACS immunomagnetic separation (IMS) system for concentrating Cryptosporidium parvum. Oocysts were first labeled with fluorescein isothiocyanate (FITC) or rabbit anti-C. parvum antibodies, then linked to MicroBeads coated with anti-FITC or anti-rabbit IgG, and separated through a high gradient separation column. Results indicated that over 95% of oocysts were recovered and their fluorescence and infectivity were retained. The presence of MicroBeads showed no effect on genomic DNA extraction and subsequent polymerase chain reaction (PCR)-based analyses, as sensitivity of PCR (10 oocysts) and the band pattern of randomly amplified polymorphic DNA (RAPD) were identical to those using DNAs extracted from normally purified oocysts. IMS-PCR consistently detected as few as 10 oocysts from 100 ml of apple juice or homogenized milk and IMS-IFA could detect 100 oocysts from 1 g of deer manure, demonstrating the efficiency of IMS in recovering oocysts from environmental and food samples. Our results suggest that the MACS IMS system could be used for multiple applications in Cryptosporidium research.