Observations on Eimeria tenella in Feces of Inoculated Chickens

SYNOPSIS. In studies on coccidial excystation, Eimeria tenella sporulated oocysts were fed to 5 individually caged chickens, and during the next 4.5 hours, all droppings were collected immediately after excretion, mixed with 0.85% NaCl solution, then promptly examined for the parasites. Some samples were placed in cold storage at 4 C and examined at intervals thereafter. Three of the birds were necropsied after 5 hours and the intestinal contents examined. Apparently unbroken oocysts containing active sporozoites were in chicken droppings beginning 1–2.25 hours postinoculation; some activated forms were inside and others outside the sporocysts. Free sporozoites, sometimes numerous, first appeared in the feces 1.1–2 hours postinoculation. At necropsy, the state and condition of the parasites in the lumen of bird intestines were similar to those in its last fecal dropping. After being in cold storage for 48 hours, free sporozoites inside and outside the oocysts became active and moved about when placed on the warming stage of the microscope.     

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.     

Susceptibility of 1- and 3-Day-Old Chicks to Infection with the Coccidium, Eimeria acervulina

SYNOPSIS. One-day-old chicks were less susceptible to experimental infection with E. acervidina than were 3-day-old chicks. Chicks fed intact oocysts when 3 days old produced 5.3, 6.7, and 42.7 times as many oocysts and had more extensive lesions than did those fed a similar number of oocysts when 1 day old. When oocyst suspensions that contained both liberated sporocysts and intact oocysts were administered, chicks infected when 3 days old produced only 1.8, 1.3, and 2.6 times as many oocysts as did those infected when 1 day old.
Examination of gizzard and intestinal contents of chicks killed 2–1½ hr after receiving massive numbers of intact oocysts showed that only a few sporocysts were liberated from oocysts in the gizzard of 1-day-old chicks, whereas more were liberated in the gizzard of 3-day-old chicks. Very few sporozoites were found in the duodenum of the 1-day-old chicks. but there was a linear increase in the percentages in samples from lower levels of the small intestine. In 3-day-old chicks, excystation in the duodenum was high and, instead of increasing, remained at about the same level in the jejunum.
The far smaller number of liberated sporocysts in the gizzards of 1-day-old chicks is attributed to less musculature and an incompletely developed grinding surface The delayed excystation of sporozoites in the intestine of 1-day-old chicks is thought to be due to suboptimal concentrations of trypsin and/or other pancreatic enzymes effecting excystation.
The lighter infections observed in 1-day-old chicks, as compared to those in chicks 3 days old, are attributed to (a) a smaller number of liberated sporocysts leaving the gizzard, (b) delayed excystation in the intestine, and (c) less opportunity for sporozoites to penetrate epithelial cells.     

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.     

Comparative Excystation of Four Species of Poultry Coccidia

SYNOPSIS. Examination of the crop, gizzard, and intestinal contents of chickens fed suspensions of either Eimeria acervulina or E. tenella oocysts and turkeys fed either E. meleagrimitis or E. gallopavonis oocysts indicated that, in all 4 species, (1) oocysts apparently remained unchanged while in the crop, (2) sporocysts were liberated from oocysts while the latter were passing through the gizzard, (3) sporozoites were activated and escaped from liberated sporocysts after they had reached the small intestine, and (4) sporozoites within intact oocysts in the crop, gizzard, and intestines were not activated.
In vitro , trypsin 1–300 alone caused a small percentage of sporozoites to excyst from mechanically liberated sporocysts. The percentage of excystation increased greatly when trypsin was added to sodium taurocholate and increased even more when it was combined with chicken or turkey bile.
The two duodenal species ( E. acervulina and E. meleagrimitis ) differed both in vivo and in vitro from the two cecal species ( E. tenella and E. gallopavonis ). The duodenal species excysted in less time and farther anteriorly in the small intestine than did the cecal species. In addition, sporozoites of the two cecal species survived much longer in media containing trypsin plus bile or sodium taurccholate than did those of the two duodenal species.     

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 Effects of Heat and Cobalt-60 Gamma-Radiation on Excystation of the Rat Coccidium,Eimeria nieschulzi Dieben, 1924*

SYNOPSIS. Sporulated oocysts of Eimeria nieschulzi Dieben, a rat coccidium, were exposed for 1 hr to Cobalt-60 γ-radiation (15, 30. or 60 k-rads), to heat (35, 40, or 45 C). or to both concurrently (15, 30, or 60 k-rads at 35 C) to compare the excystation capabilities of treated vs nontreated parasites. Intact, treated oocysts appeared structurally unaltered when viewed with the light microscope. Excystation of sporozoites occurred in all treated groups when their sporocysts were exposed to a trypsin-sodium taurocholate (TST) fluid, but after 150 min in TST the excystation rate was significantly lower than in non-treated sporocysts. Sporozoites which excysted from treated sporocysts were abnormal both in the excystation process and in their form and movement once outside the sporocyst.     

Eimeria crotalviridis sp. n. from Prairie Rattlesnakes, Crotalus viridis viridis, in New Mexico with Data on Excystation of Sporozoites and Ultrastructure of the Oocyst Wall

SYNOPSIS Oocysts of Eimeria crotalviridis sp. n. are described from prairie rattlesnakes, Crotalus viridis viridis in New Mexico on the basis of light and electron microscopy and in vitro excystation of sporozoites. Sporulated oocysts of E. crotalviridis are elliptical, 26.4 × 22.3 (23–29 × 20–24) μm with ovoid sporocysts 11.7 × 8.1 (11–13 × 7–9) μm. A micropyle, micropyle cap and polar bodies are absent, but oocyst and sporocyst residua and Stieda and substieda bodies are present. Excysted sporozoites are 12.4 × 2.8 (11–13 × 2–3) μm and have 1 large posterior refractile body and a nucleus with a prominent nucleolus. Ultrastructurally, the oocyst wall has 2 layers, a thick, electron-dense, highly sculptured outer layer composed of a fine granular matrix and a thin, granular, osmiophilic inner layer, separated from the outer layer by at least one unit membrane. These layers are 441 (353–510) and 21.6 (19–29) nm thick, respectively. Within 15 min after exposure to a trypsin-sodium taurocholate fluid, sporozoites of E. crotalviridis excysted from 5-month-old sporocysts.     

Purification of Eimeria Sporozoites by DE-52 Anion Exchange Chromatography

An anion exchange column of DE-52 has been used to purify Eimeria sporozoites from a post-excystation mixture of oocysts, oocyst shells, sporocysts, sporocyst shells, and sporozoites. The mean recovery from several experiments was 94% and virtually all non-sporozoite material was removed. Infectivity studies in vitro with sporozoites showed that they were viable after purification and were at least as infectious as the unpurified sporozoites; furthermore, oocysts in the crude preparation could be recovered from the DE-52 cellulose by resuspending them in a 20% (w/v) sodium chloride solution.     

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.     

Optimized excystation protocol for ruminant Eimeria bovis- and Eimeria arloingi-sporulated oocysts and first 3D holotomographic microscopy analysis of differing sporozoite egress

Successful excystation of sporulated Eimeria spp. oocysts is an important step to acquire large numbers of viable sporozoites for molecular, biochemical, immunological and in vitro experiments for detailed studies on complex host cell-parasite interactions. An improved method for excystation of sporulated oocysts and collection of infective E. bovis- and E. arloingi-sporozoites is here described. Eimeria spp. oocysts were treated for at least 20 h with sterile 0.02 M _L-cysteine HCl/0.2 M NaHCO₃ solution at 37 °C in 100% CO₂ atmosphere. The last oocyst treatment was performed with a 0.4% trypsin 8% sterile bovine bile excystation solution, which disrupted oocyst walls with consequent activation of sporozoites within oocyst circumplasm, thereby releasing up to 90% of sporozoites in approximately 2 h of incubation (37 °C) with a 1:3 (oocysts:sporozoites) ratio. Free-released sporozoites were filtered in order to remove rests of oocysts, sporocysts and non-sporulated oocysts. Furthermore, live cell imaging 3D holotomographic microscopy (Nanolive®) analysis allowed visualization of differing sporozoite egress strategies. Sporozoites of both species were up to 99% viable, highly motile, capable of active host cell invasion and further development into trophozoite- as well as macroment-development in primary bovine umbilical vein endothelial cells (BUVEC). Sporozoites obtained by this new excystation protocol were cleaner at the time point of exposure of BUVEC monolayers and thus benefiting from the non-activation status of these highly immunocompetent cells through debris. Alongside, this protocol improved former described methods by being is less expensive, faster, accessible for all labs with minimum equipment, and without requirement of neither expensive buffer solutions nor sophisticated instruments such as ultracentrifuges.     

The Structure of the Oocyst and the Excystation Process of Isospora marquardti sp. n. from the Colorado pika, Ochotona princeps

SYNOPSIS. Oocysts of Isospora marquardti sp. n. from the Colorado pika, Ochotona princeps , have spheroid oocysts, 30.5 (23–36) μm in diameter, and ovoid sporocysts, measuring 19.3 × 12.0 (17–22 × 10–14) μm. A polar body, 2 × 4 μm, a spheroid sporocyst residuum (8.3 μm in diameter), a Stieda body, and a distinct substiedal body (3 × 3 μm) are present. A micropyle and an oocyst residuum are absent. Excysted sporozoites, averaging 3.0 × 18.5 (2–4 × 15–20) μm, contain 2 refractile globules, 1 on each side of the nucleus with a prominent nucleolus.
The sporozoite excystation process using a trypsin-sodium taurocholate fluid is described.     

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.     

Pancreatic chymotrypsin as the essential enzyme for excystation of Eimeria tenella.

Sporocysts from the protozoan parasite, Eimeria tenella, were isolated, preincubated with sodium taurocholate, and treated with various preparations of pancreatic enzymes. Crude trypsin, crude lipase, and purified alpha-chymotrypsin all could break the shells of sporocysts and release sporozoites. Purified trypsin was much less active than crude trypsin and purified lipase showed no activity at all. Specific inhibitors of chymotrypsin, tosyl-L-phenylalanyl chloromethane, diphenylcarbamyl chloride, and chymostatin inhibited the release of sporozoites by all the enzyme samples, whereas tosyl-L-lysyl chloromethane, a specific inhibitor of trypsin, exerted no inhibitory effect. It is thus postulated that chymotrypsin, not trypsin, is an essential enzyme involved in excystation of E. tenella. Purified chymotrypsin is recommended to replace crude trypsin in the vitro excystation of E. tenella as a likely improved procedure.     

Description of Pythonella scleruri n. sp. (Apicomplexa: Eimeriidae) from a brazilian bird rufous-breasted-leaftosser Sclerurus scansor, 1835 (Passeriformes: Furnariidae)

Coccidian oocysts containing 16 sporocysts with 4 sporozoites in each were observed in a faecal sample from Sclerurus scansor collected in the Itatiaia National Park, southeastern region of Brazil. The oocysts are characterized by ellipsoidal shape measuring 42.5 x 32.8 mm, with smooth, thick double-layered wall of a greenish-orange colour. An oocyst residuum of numerous scattered granules among the sporocysts in sporulated ones; 16 round sporocysts, averaging 10.5 x 10 mm each containing four elongated sporozoites; presence of residuum; absence of Stieda body. The presently described coccidian, recorded for the first time in birds, is a new species named P. scleruri.     

Early events in the life cycle of the mouse coccidium Eimeria falciformis (Eimer, 1870) Schneider, 1875 in naive and immune hosts

The initial phases of invasion of mammalian coccidia of the genus Eimeria into host tissue are still poorly known. This process, including the passage of oocysts through the intestinal lumen, excystation of sporozoites, their penetration into epithelial cells and migration to the target site was studied in both naive and immune mice infected with Eimeria falciformis. After oral infection, the intact oocysts were transported with enteral contents to the large intestine, where the excystation of sporozoites and their penetration into superficial epithelium took place. The sporozoites subsequently migrated into the epithelium of crypts, which is the specific site of asexual multiplication. The immune status of the hosts did not affect the passage of oocysts, excystation and penetration of sporozoites. However, the migration of sporozoites towards their target site (crypts) was impeded in immune mice and sporozoites tended to remain in superficial mucosa rather than migrate to the crypts.     

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.     

Isolation of purified oocyst walls and sporocysts from Toxoplasma gondii

Toxoplasma gondii oocysts are environmentally resistant and can infect virtually all warm-blooded hosts, including humans and livestock. Little is known about the biochemical basis for this resistance of oocysts, and mechanism for excystation of T. gondii sporozoites. The objective of the present study was to evaluate different methods (mechanical fragmentation, gradients, flow cytometry) to separate and purify T. gondii oocyst walls and sporocysts. Oocyst walls were successfully separated and purified using iodixanol gradients. Sporocysts were successfully separated and purified using iodixanol and Percoll gradients. Purification was also achieved by flow cytometry. Flow cytometry with fluorescence-activated cell sorting (FACS) yielded analytical quantities of oocyst walls and intact sporocysts. Flow cytometry with FACS also proved useful for quantitation of purity obtained following iodixanol gradient fractionation. Methods reported in this paper will be useful for analytical purposes, such as proteomic analysis of components unique to this life cycle stage, development of detection methods, or excystation studies.     

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.