The Effects of Reducing Conditions, Medium, pH, Temperature, and Time on in Vitro Excystation of Cryptosporidium

ABSTRACT. Whereas excystation of sporozoites from oocysts of most coccidian species requires exposure to reducing conditions followed by pancreatic enzymes and bile salts, sporozoites of a bovine isolate of Cryptosporidium excysted without exposure to either reducing conditions or to pancreatic enzymes and bile salts. Without prior exposure to reducing conditions, a high percent excysted after incubation in a mixture of trypsin and bile salts in Ringer's solution; fewer excysted after incubation in tap water, even fewer after incubation in salt solutions, and none after incubation in saliva. Excystation, generally greater at pH 7.6 than at pH 6.0 and at 37°C than at 20°C, was observed as early as 1 h after incubation in water or the trypsin-bile mixture. These findings provide circumstantial evidence that oocysts of Cryptosporidium can excyst in extraintestinal sites and liberate sporozoites that can initiate autoinfection.     

The Effects of pH,Buffers, Bile and Bile Acids on Excystation of Sporozoites of Various Eimeria Species*

SYNOPSIS. Oocysts of Eimeria bovis were found to undergo excystation when subjected at 39 C to a pretreatment consisting of exposure for 24 hr to CO₂ and air (50–50), and a treatment for 7 hr with a mixture of bile and trypsin. At pH's of 6.0 thru 10.0 with tris-maleate buffer, excystation occurred over the entire range of pH tested, with the highest levels at pH 7.5-8.5. No adverse or inhibitive effect on excystation or the viability of the sporozoites was observed. Disintegration of sporozoites occurred within the sporocysts of intact oocysts at each of the pH levels studied when boric acid-borax, ammediol, and glycine-sodium hydroxide buffers were used in the treatment medium. Phosphate buffer inhibited excystation when used in the excysting medium. Excystation occurred at levels above 90% in all dilutions of taurocholic, glycocholic, glycotaurocholic, and cholic acids included in the study (0.5-10.0%) except for the 10% and 5% dilutions of cholic acid and the 10% dilution of glycotaurocholic acid. In the latter 3 dilutions, sporozoites within the sporocysts of intact oocysts disintegrated. Excystation levels above 90% were observed in the 50% and 10% dilutions of fresh bovine bile, and in the 5% dilution of lyophilized bovine bile. Lower levels of excystation occurred in greater dilutions of both kinds of bile. No excystation occurred when any of the bile acids, fresh bovine bile or lyophilized bile were used without trypsin, except for fresh bile that contained a heavy suspension of bacteria and fungi. In a medium containing trypsin and heat-treated bile, heat-treated bile acids, or no bile, 2.5–8% of the oocysts excysted. The findings indicate that satisfactory excystation can be obtained with a treatment medium containing tris-maleate at pH 7.5–8.5, 0.25% trypsin, and 1% of one of the bile acids.     

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.     

The utilization of sodium taurocholate in excystation of Cryptosporidium parvum and infection of tissue culture.

The present work deals with optimization of excystation of Cryptosporidium parvum oocysts and the infection process of tissue culture cells by the parasite. It was shown that presence of the bile salt sodium taurocholate in the incubation medium expedited excystation of the tested GCH1 isolate and enhanced it, as compared with bleaching of the oocysts. This bile salt had no effect on the viability of tissue culture cell lines MDBK and HCT-8 at the tested concentration of 0.375% for up to 2 hr of coincubation. Infection studies conducted on tissue culture cells showed higher infection rates in the presence of sodium taurocholate than with bleached oocysts in the absence of this bile salt. It may be concluded that, at least as regards the GCH1 strain of C. parvum, the whole infection process can be performed in the presence of sodium taurocholate, and does not require separation and cleaning of the excysted sporozoites before their application to tissue culture cells.     

Chemical and physical factors affecting the excystation of Cryptosporidium parvum oocysts.

Cryptosporidium parvum oocysts were examined to ascertain excystation requirements and the effects of gamma irradiation. Oocysts and excysted sporozoites were examined for dye permeability and infectivity. Maximum excystation occurred when oocysts were pretreated with acid and incubated with bile salts, and potassium or sodium bicarbonate. Pretreatment with Hanks' balanced salt solution or NaCl lowered excystation; however, this effect was overcome with acid. Sodium ions were replaceable with potassium ions, and sodium bicarbonate was replaceable with sodium phosphate. Oocysts that received 200 krad irradiation excysted at the same rates as nonirradiated oocysts (95%), the excystation rates were lowered (50%) by 2,000 krad, and no excystation was observed by 5,000 krad. No differences were observed between the propidium iodide (PI) permeability of untreated oocysts and oocysts treated with 200 krad, while 92% of oocysts were PI positive after 2,000 krad. Most of the sporozoites exposed to 2,000 krad were not viable as indicated by the dye permeability assay. The oocysts irradiated with 200 and 2,000 krad infected cells, but no replication was observed. The results suggest that gamma-irradiated oocysts may still be capable of excystation and apparent infection; however, because the sporozoites could not reproduce they must not have been viable.     

In vitro excystation of Cryptosporidium baileyi from chickens

Release of sporozoites from the oocysts of Cryptosporidium baileyi is described from Nomarski interference-contrast microscopy. Just prior to excystation, the four sporozoites became motile and rearranged themselves within the oocyst. The sporozoites were then rapidly expelled through an opening that formed in the oocyst wall, and the residuum was either released or retained within the oocyst. Excysted sporozoites were crescent shaped and measured 5.0-9.0 microns X 1.0-1.6 micron (mean = 6.8 X 1.1 microns). Excystation occurred when sodium taurocholate or a mixture of trypsin and sodium taurocholate was present in the incubation medium. High levels of excystation occurred at 37 degrees or 40 degrees C, but excystation did not occur at 4 degrees C. The ability of biles from two avian and two mammalian hosts to produce excystation of C. baileyi was also studied. After a 2-h incubation at 40 degrees C, the percentages of excystation were 69.5% in goat bile, 45.0% in pig bile, 33.0% in chicken bile, and 34.5% in turkey bile.     

Excystation of the Poultry Coccidium, Eimeria acervulina

SYNOPSIS. Using intervals up to 5 hours, attempts to excyst sporozoites of Eimeria acervulina from intact oocysts in vitro were unsuccessful.
Examination of crop, gizzard, and intestinal contents of chicks fed large numbers of sporulated oocysts indicated that (1) no obvious change in the oocysts occurred in the crop, (2) a high percentage of the sporocysts were quickly released from the oocysts in the gizzard, (3) the sporozoites escaped from the liberated sporocysts in the duodenum and jejunum, and (4) the action of the digestive juice was apparently on the sporocysts rather than on the oocysts.
In vitro attempts to excyst sporozoites from free sporocysts with various pancreatic preparations in the absence of bile produced low or insignificant percentages of excystation. In the presence of bile, bile salts, and other surface-active agents, the action of the pancreatic preparations was greatly increased. The heaviest suspension of motile, nonaggregating sporozoites was obtained with 0.25% trypsin 1–300 in 5% chicken bile at pH 7.6.     

Proteomics analysis and protein expression during sporozoite excystation of Cryptosporidium parvum (Coccidia, Apicomplexa)

Cryptosporidiosis, caused by coccidian parasites of the genus Cryptosporidium, is a major cause of human gastrointestinal infections and poses a significant health risk especially to immunocompromised patients. Despite intensive efforts for more than 20 years, there is currently no effective drug treatment against these protozoa. This study examined the zoonotic species Cryptosporidium parvum at two important stages of its life cycle: the non-excysted (transmissive) and excysted (infective) forms. To increase our understanding of the molecular basis of sporozoite excystation, LC-MS/MS coupling with a stable isotope N-terminal labeling strategy using iTRAQ reagents was used on soluble fractions of both non-excysted and excysted sporozoites, i.e. sporozoites both inside and outside oocysts were examined. Sporozoites are the infective stage that penetrates small intestinal enterocytes. Also to increase our knowledge of the C. parvum proteome, shotgun sequencing was performed on insoluble fractions from both non-excysted and excysted sporozoites. In total 303 C. parvum proteins were identified, 56 of which, hitherto described as being only hypothetical proteins, are expressed in both excysted and non-excysted sporozoites. Importantly we demonstrated that the expression of 26 proteins increases significantly during excystation. These excystation-induced proteins included ribosomal proteins, metabolic enzymes, and heat shock proteins. Interestingly three Apicomplexa-specific proteins and five Cryptosporidium-specific proteins augmented in excysted invasive sporozoites. These eight proteins represent promising targets for developing vaccines or chemotherapies that could block parasite entry into host cells.     

The use of sodium taurodeoxycholate for excystation of Eimeria tenella sporozoites

As an in vitro excystor, sodium taurodeoxycholate (TDC) released 80--90% Eimeria tenella sporozites, in contrast to 0--15% excystation by six other bile salts or bile extracts, and pooled chicken bile in 90 min at 37 C with continuous agitation. Pooled chicken bile required 4 to 4 1/2 hr to excyst similar percentages of sporozoites. Prolonged incubation with other bile salts and bile extracts excysted most sporozoites, but killed them. When the incubation temperature was raised to 44 C, TDC excysted 100% of the sporozoites in 60 min. In all other bile salts or bile extracts, the percentage of excystation increased greatly at 44 C, but none equalled that of TDC. The molecular similarity of TDC to a naturally occurring bile salt of chickens is presented as an explanation for the superior performance of TDC as an excystor. Data are examined to minimize the possibility that excysting activity of TDC can be attributed to other bile salts present at impurities.     

Carbon Dioxide as the Initial Stimulus for Excystation of Eimeria tenella Oocysts*

SYNOPSIS. The stimulus necessary to initiate in vitro excystation of the chicken coccidium Eimeria tenella was provided by exposure of intact sporulated oocysts to an atmosphere of carbon dioxide. This stimulus produced a thinning and indentation at the micropylar region and oocysts became permeable to trypsin and bile. Sporozoites became active and began to escape from sporocysts into the oocyst cavity and then to the outside thru the altered micropyle after incubation in the enzyme-bile mixture. Activation of sporozoites when CO₂-pretreated oocysts were incubated in trypsin and bile, was used as the criterion to determine the number of oocysts responding to the initial stimulus. Thus, activation of sporozoites within intact oocysts was an indirect measurement of the number of oocysts stimulated during CO₂-pretreatment. Approximately 90% of the oocysts contained active sporozoites after 18 hr of pretreatment with carbon dioxide and 8 hr incubation in trypsin and bile at 38 or 41 C, respectively. Pretreatment of oocysts with air, N₂, O₂, or He resulted in 8% or less activation during incubation in trypsin and bile. Approximately 83% of the oocysts responded to the stimulus during 8 hr CO₂-pretreatment at 41 C, whereas at 38 C, 16 hr of pretreatment were required for a similar response. The stimulus did not elicit a response from oocysts held at 23 C during the pretreatment gasphase. No significant difference occurred in number of oocysts containing active sporozoites after sufficient CO₂-pretreatment for maximum stimulation of oocysts and incubation in trypsin and bile at 38 or 41 C.     

A simple and reliable method of producing in vitro infections of cryptosporidium parvum (apicomplexa)

Abstract A variety of techniques have been used to infect cell monolayers in culture with the protozoan, Cryptosporidium parvum . However, most of these methods rely on the use of trypsin and/or bile salts to excyst sporozoites in vitro, followed by washing sporozoites free of excystation solution prior to their addition to subconfluent monolayers. This method not only increases the amount of time required to establish infections in vitro, but also results in prolonged exposure of free sporozoites to environmental conditions. Here we report a simple, fast, and efficient method of obtaining consistent infections of C. parvum in cell monolayers. This technique relies on the ability of the parasite to excyst at 37°C but not at room temperature following pretreatment with sodium hypochlorite. By adding surface-sterilized oocysts directly to monolayers, sporozoites have access to host cells immediately upon excystation.     

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.     

In Vitro Excystation of Cryptosporidium baileyi from Chickens1

Release of sporozoites from the oocysts of Cryptosporidium baileyi is described from Nomarski interference-contrast microscopy. Just prior to excystation, the four sporozoites became motile and rearranged themselves within the oocyst. The sporozoites were then rapidly expelled through an opening that formed in the oocyst wall, and the residuum was either released or retained within the oocyst. Excysted sporozoites were crescent shaped and measured 5.0–9.0 μm × 1.0–1.6 μm (x?= 6.8 × 1.1 μm). Excystation occurred when sodium taurocholate or a mixture of trypsin and sodium taurocholate was present in the incubation medium. High levels of excystation occurred at 37° or 40°C, but excystation did not occur at 4°C. The ability of biles from two avian and two mammalian hosts to produce excystation of C. baileyi was also studied. After a 2-h incubation at 40°C, the percentages of excystation were 69.5% in goat bile, 45.0% in pig bile, 33.0% in chicken bile, and 34.5% in turkey bile.     

In Vitro Excystation of Caryospora simplex (Apicomplexa: Eimeriorina)1

In vitro excystation of sporozoites of the heteroxenous coccidian Caryospora simplex Léger, 1904 (Apicomplexa: Eimeriorina) is described. Sporocysts freed mechanically from oocysts released a maximum of 51% of their sporozoites within 45 min at 25°C and a maximum of 74% within 20 min at 37°C when incubated in a 0.25% (w/v) trypsin–0.75% (w/v) sodium taurocholate (bile salt) excystation solution. At emergence from sporocysts, sporozoites were weakly motile then became highly active after about 2 min in excystation solution. Sporozoites within sporocysts exposed to bile salt only became highly motile within 25 min at 25°C and within 15 min at 37°C but did not excyst. When exposed only to trypsin at the above temperatures, the Stieda body dissolved; the substieda body remained intact, and the sporozoites exhibited only limited motility within sporocysts; only a few excysted. Intact, sporulated oocysts incubated at 25° or 37°C in 0.02 M cysteine-HC1 and a 50% CO₂ atmosphere for 18 h had no morphologic changes in the oocyst wall. Further incubation of these intact oocysts in excystation solution for 30 min at 37°C caused neither motility of sporozoites within sporocysts nor excystation. Grinding oocysts for 30 sec in a motor-driven, teflon-coated tissue grinder caused motility of some sporozoites within sporocysts but did not result in excystation.     

In vitro excystation of Sarcocystis capracanis, Sarcocystis cruzi and Sarcocystis tenella (Apicomplexa)

Improved rates of in vitro excystation of sporozoites from sporocysts of Sarcocystis capracanis, Sarcocystis cruzi, and Sarcocystis tenella were obtained by pretreating sporocysts with an aqueous sodium hypochlorite (NaOCl) solution followed by incubation in excysting fluid (EF). After pretreatment with NaOCl, sporocysts were washed 4 times in Hanks' balanced salt solution and then incubated in various EF (pH 7.4) at 38.5 C in 5% CO2-95% air. Maximum rates of excystation (free sporozoites/(sporozoites in sporocysts + free sporozoites) X 100) for all 3 species of Sarcocystis occurred at 4 hr after incubation in EF. These rates were 17% for S. capracanis after incubation in EF containing 2% trypsin + 10% caprine bile; 90% for S. cruzi in 2% trypsin + 10% bovine bile; and 20% for S. tenella in 2% trypsin + 10% caprine bile. Only a 40% excystation rate occurred in sporocysts of S. cruzi that had been stored previously for 14 days in aqueous potassium dichromate. Excysted sporozoites of S. capracanis, S. cruzi, and S. tenella penetrated and developed to mature meronts in bovine pulmonary artery endothelial cells or bovine monocytes.     

An improved method for the analysis of Cryptosporidium parvum oocysts by matrix-assisted laser desorption/ionization time of flight mass spectrometry

Cryptosporidium parvum oocysts were analyzed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Sample preparation proved to be a crucial step in the acquisition of acceptable mass spectra. Oocysts of C. parvum and the matrix were mixed and held for at least 45 min to produce reproducible, representative mass spectra. Sporozoites were also excysted from oocysts, purified, and analyzed using MALDI-TOF MS. The mass spectra of the intact oocysts contained many of the same peaks found in the mass spectra of the sporozoites, suggesting that during analysis, the internal constituents, not just the oocyst wall, are ablated by the laser.     

Effects of Carbon Dioxide on Coccidian Oocysts from 6 Host Species*

SYNOPSIS Activation of sporozoites in oocysts of Eimeria acervulina (chicken), E. intricata (sheep), and E. scabra (swine) occurred after pretreatment in aqueous 0.02 M cysteine hydrochloride under an atmosphere of CO₂, followed by incubation in a trypsin-bile mixture. Sporozoites of E. stiedae (rabbit), E. bilamellata (squirrel), and Isospora canis (dog) became activated when incubated in trypsin and bile with or without prior CO₂-pretreatment of oocysts; however, when CO₂-pretreatment was used, activation of these species in trypsin and bile was greatly enhanced. For E. acervulina, 12% of the oocysts were activated after 4 hr CO₂-pretreatment and 10 hr incubation in trypsin and bile at 43 C; higher temperatures or longer pretreatment times did not cause greater activation. Eimeria intricata oocysts became activated after 1 hr pretreatment and 10 hr incubation in trypsin and bile at 37, 39 or 41 C, respectively. The highest activation (31%) occurred after 20 hr pretreatment and 10 hr incubation in trypsin and bile at 41 C. Ninety percent of E. scabra oocysts contained active sporozoites after 1 hr CO₂-pretreatment and 10 hr incubation in trypsin and bile at 37 C. At 39 or 41 C, 100% activation occurred with this species after similar pretreatment and treatment periods. With E. bilamellata, 64% activation occurred in nonpretreated oocysts incubated 10 hr in trypsin and bile at 41 C, whereas 100% activation occurred if oocysts were pretreated with CO₂ for 1 hr before treatment with trypsin and bile. Thirty-one, 35, and 36% of CO₂-pretreated E. stiedae oocysts were activated after 1 hr incubation in trypsin and bile at 37, 39 or 41 C, respectively, whereas 1, 2, and 20% activation occurred in nonpretreated oocysts incubated at the same temperatures. Sporozoites in 99-100% of I. canis oocysts were activated after 10 hr treatment in trypsin and bile with or without 1 hr CO₂-pretreatment at 23, 37, 39 or 41 C.     

Massive excystation of Toxoplasma gondii sporozoites

In the present study, several stimuli were tested to liberate Toxoplasma gondii sporozoites from oocysts. Incubation in Na(2)CO(3)-CO(2) buffer at 40 degrees C with ultrasound, followed by incubation at 40 degrees C with bile or deoxycholic acid, was the only method that resulted in 95% liberation of motile infective sporozoites. A protocol of reproducibly high efficiency is provided, that compares favorably with any of the methods previously published.     

Complete development of Cryptosporidium parvum in MDBK cells

Abstract Sporozoites of Cryptosporidium parvum excysted in vitro from bovine oocysts were incubated with monolayers of Madin-Darby bovine kidney cells. The extent of parasite colonisation was monitored by light microscopy and immunofluorescence. Electron microscopy confirmed the complete development and replication of C. parvum within Madin-Darby bovine kidney cells.     

Studies on cryopreservation of Cryptosporidium parvum

Neonatal BALB/c mice received oocysts or sporozoites of Cryptosporidium parvum pretreated by a variety of cryopreservation protocols. Histologic sections of infected and control mice were examined to determine if pretreated organisms established infection in the intestine. Sporozoites were inoculated rectally, oocysts orally. Freshly excysted sporozoites were frozen in Hanks' balanced salt solution (HBSS) containing dimethylsulfoxide (DMSO) or glycerol at concentrations of 5%, 10%, or 15% at cooling rates of -1 C and -10 C per min. Other sporozoites were frozen to -70 C in the absence of cryoprotectant without controlled reduction of temperature, others placed in HBSS with 10% DMSO but not subjected to freezing, whereas others were placed in vitrification media containing 5.5 M propylene glycol, 6.5 M glycerol, or 8 M ethylene glycol for 1 min before resuspension in fresh HBSS and inoculation into mice. Intact oocysts were frozen without controlled reduction of temperature directly to -70 C in HBSS containing no cryoprotectant or in HBSS that contained 10% DMSO. Others were cooled at -0.3 C per min from 4 C to -70 C in HBSS with 5% or 10% DMSO. Still others were cooled at a rate of -1 C per min until reaching -40 C and then cooled at -10 C per min until reaching -70 C in HBSS with 7.5% DMSO. Oocysts and sporozoites not exposed to cryoprotectants were inoculated into mice orally and rectally, respectively, for control purposes. Only unfrozen oocysts and sporozoites not exposed to cryoprotectant, and some of the unfrozen oocysts and sporozoites exposed to 10% DMSO, successfully established infections in mice.