Eimeria maxima (Apicomplexa): a comparison of sporozoite transport in naive and immune chickens

This study compared the early stages of infection in naive and immune chickens infected with Eimeria maxima. An immunoperoxidase stain was developed and used to detect sporozoites and early schizonts in tissue sections of intestinal epithelium. A significantly higher proportion of sporozoites was present in the crypts of naive chickens, 48 hr postinoculation of oocysts, compared to immune chickens. Sporozoites in immune birds tended to remain in the lamina propria rather than migrate to the crypts. Sporozoites were found within intraepithelial lymphocytes (IEL's) in the epithelium, the lamina propria, and the crypts of both naive and immune chickens. Parasites in IEL's of immune birds at the ultrastructural level and there were no apparent morphological abnormalities. Livers and spleens, of both immune and naive chickens that had been inoculated with Eimeria maxima, produced patent infections when fed to susceptible chickens. Infections could be transferred up to 72 hr post-inoculation of the donor birds. Peak oocyst production in the recipient birds occurred 7-8 days after the transfers. This time period approximates the prepatent period in a natural infection and thus implies that the extraintestinal stage was a sporozoite.     

Excystation of the Sporozoites of Eimeria tenella in Apparently Unbroken Oocysts in the Chicken

SYNOPSIS. Chickens experimentally fed sporulated oocysts of Eimeria tenella were necropsied 5–300 minutes later to study excystation, especially in apparently unbroken oocysts. In birds killed 75–125 minutes after inoculation, there was evidence of excystation in some oocysts recovered from the small and large intestines. Altho not visibly broken, the shells of about 10% of the ones studied were wrinkled or otherwise deformed; many contained active sporozoites, some inside and some outside the sporocysts. During examinations, numerous sporozoites emerged from the sporocysts. Altho evidence was not obtained that excystation from unbroken oocysts was occurring inside the birds, the fact that it occurred in some oocysts during examinations may indicate that it could be taking place. Evidence of excystation was not seen in oocysts with shells that were not visibly altered.     

Storage Polysaccharide in Coccidial Sporozites after Excystation and Penetration of Cells

SYNOPSIS. Eimeria acervulina, E. necatrix , and E. meleagrimitis sporozoites were examined for carbohydrates by cytochemical methods during dormancy, after excystation, and after penetration of cells. The only carbohydrate found was amylopectin, a homogeneous polymer of glucose. It was distributed in 3 regions: (a) in front of the anterior refractile globule, (b) around the nucleus, and (c) behind the posterior refractile globule. The relative amounts decreased after excystation and penetration of cells until only small amounts remained around the nucleus. The quantity of amylopectin decreased following excystation from 30.0-36.7 to 9.4-13.3 μg glucose/10⁶ oocysts. Over a 6 yr period of storage at 4 C, there was a decrease in the quantity of amylopectin in dormant sporozoites of E. acervulina from 33.3 μg glucose/10⁶ oocysts at 3 mos to 1.5 μg at 6 years. Coincidentally, 3 month- and 1 year-old oocysts of E. acervulina produced patent infections in chicks with a dosage of 5 × 10⁴ oocysts, but only a few of the oocysts that had been stored for 2 years were infective; a dosage of 2 × 10⁶ oocysts was necessary to produce a patent infection. Oocysts which had been stored 6 years did not produce a patent infection.
It was concluded that amylopectin is the energy source for excystation and subsequent penetration of cells. Small amounts of amylopectin are used during dormancy and, when the content in the sporozoite falls below a certain level, the sporozoites lack sufficient energy to infect cells.     

Effects of intestinal contents from normal and immunized mice on sporozoites of Eimeria falciformis

The interaction of Eimeria falciformis sporozoites with the intestinal epithelium and with the intestinal contents from the cecum and colon of normal and specifically immunized mice was studied by light (LM) and scanning electron (SEM) microscopy. Fecal (FM) and enterocyte-associated (EAM) mucus were removed from the cecum and colon of normal mice and mice that had been immunized 1, 6, 12, or 20 days earlier with a series of oral inoculations of E. falciformis oocysts. Sporozoite-specific IgA, but neither IgM nor IgG, was detected by the immunofluorescent antibody test in FM and EAM from immunized mice. No sporozoite-specific immunoglobulin was detected in normal mice. When examined by LM, sporozoites exposed to all FM and EAM preparations exhibited greater motility and excystation from sporocysts. At 4 h after incubation in FM or EAM from normal or immune mice, about 10% of the sporozoites appeared damaged, being non-motile and non-refractile. Immune FM and EAM caused agglutination of sporozoites and sporocysts and oocyst walls of E. falciformis, but did not agglutinate those of E. ferrisi. Scanning electron microscopy of in vitro interactions between E. falciformis sporozoites and intestinal contents revealed that sporozoites exposed to immune EAM were coated with particulate material whereas those exposed to normal EAM were relatively clean. Sporozoites exposed to immune FM were usually embedded within the mucus whereas those exposed to normal FM were situated on top of the mucus. No significant differences occurred between the length/width (L/W) ratios of sporozoites incubated in normal and EAM or in PBS.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Improved Method for High-Yield Excystation and Purification of Infective Sporozoites of Eimeria spp.1

This report describes a new, gentle procedure for rapid and efficient excystation of large numbers of infective sporozoites of Eimeria vermiformis and Eimeria stiedai. Excysted sporozoites are purified using modifications of a previously described ion-exchange chromatography method. The procedure avoids physical breakage of oocysts and results in greater than 70% recovery of the sporozoites present as sporulated oocysts (i.e. 5–6 sporozoites per sporulated oocyst). The recovered sporozoites are greater than 95% pure and are infective in vivo. We routinely isolate greater than 2 times 10⁸ sporozoites without the use of specialized or expensive equipment.     

Effects of Intestinal Contents from Normal and Immunized Mice on Sporozoites of Eimeria falciformis1

The interaction of Eimeria falciformis sporozoites with the intestinal epithelium and with the intestinal contents from the cecum and colon of normal and specifically immunized mice was studied by light (LM) and scanning electron (SEM) microscopy. Fecal (FM) and enterocyte-associated (EAM) mucus were removed from the cecum and colon of normal mice and mice that had been immunized 1, 6, 12, or 20 days earlier with a series of oral inoculations of E. falciformis oocysts. Sporozoite-specific IgA, but neither IgM nor IgG, was detected by the immunofluorescent antibody test in FM and EAM from immunized mice. No sporozoite-specific immunoglobulin was detected in normal mice. When examined by LM, sporozoites exposed to all FM and EAM preparations exhibited greater motility and excystation from sporocysts. At 4 h after incubation in FM or EAM from normal or immune mice, about 10% of the sporozoites appeared damaged, being non-motile and non-refractile. Immune FM and EAM caused agglutination of sporozoites and sporocysts and oocyst walls of E. falciformis. but did not agglutinate those of E. ferrisi. Scanning electron microscopy of in vitro interactions between E. falciformis sporozoites and intestinal contents revealed that sporozoites exposed to immune EAM were coated with particulate material whereas those exposed to normal EAM were relatively clean. Sporozoites exposed to immune FM were usually embedded within the mucus whereas those exposed to normal FM were situated on top of the mucus. No significant differences occurred between the length/width (L/W) ratios of sporozoites incubated in normal FM and EAM or in PBS. Sporozoites exposed to immune FM had significantly greater L/W ratios than those exposed to normal FM whereas those exposed to immune EAM had significantly shorter L/W ratios than ones exposed to normal EAM. Few of the sporozoites observed on the luminal surface of the colon and cecum of normal mice were covered by mucus and none was altered in shape or showed pellicular damage. Only a few sporozoites were observed on the luminal surface of the colon and cecum of immunized mice. Most of these were covered by mucus and some exhibited pellicular alterations.     

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.     

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.     

Immune responses to infections with coccidia in chickens: gut hypersensitivity.

Sporulated oocysts or oocyst hulls of Eimeria acervulina given via the crop, and oocyst juice or sporozoites inoculated directly into the intestinal lumen, were tested for their effects on the hypersensitivity of the intestines of immunized and susceptible birds. Only viable sporozoites consistently caused an increase in the permeability of the immunized intestine to macromolecules, shown by the leakage of intravenously injected dye. Repulsion of epithelium and disruption of the villi were seen more often in immunized than in susceptible chickens but there was no correlation with treatment or with dye leakage. The hypersensitive response of immune intestines challenged with a heterologous organism was approximately half as intense as the response to homologous challenge. The significance of these findings in relation to immunity to coccidiosis is discussed.     

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.     

Eimeria tenella: hsp70 expression during sporogony.

There is an increasing interest in identifying the parasite components involved in the maturation, development, and infectivity of intracellular protozoan parasites. In the present study, a heat shock protein (hsp) of the family of 70 kDa hsp (hsp70), which play important roles in the stage conversion and virulence of these parasites, was examined. Whereas hsp70 expression has been examined in Eimeria tenella within host tissues, in the present study, oocysts of E. tenella were used to investigate the expression of hsp70 during sporulation without interference from the host; hsp70 expression during excystation was induced by incubating sporulated oocysts under various experimental conditions to produce the stimuli necessary for sporozoites to become active and to excyst in vitro. Hsp70 was detected by immunohistochemical techniques; quantitative flow cytometric analysis was also been carried out using specific monoclonal antibodies against hsp70. Hsp70 was expressed during sporulation but was not found in sporulated oocysts after the completion of sporulation. Oocysts re-expressed hsp70 when excystation was induced. The presence of hsp70 prior to infection may preadapt the parasite for additional stress in the host and may be involved in the formation of sporozoites.     

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.     

Coccidia (Eimeria tachyglossi n. sp., E. echidnae n. sp., and Octosporella hystrix n. sp.) in the Echidna, Tachyglossus aculeatus (Monotremata: Tachyglossidae)

Oocysts of Octosporella hystrix n. sp., Eimeria tachyglossi n. sp., and E. echidnae n. sp. are described from the feces of the echidna Tachyglossus aculeatus (Monotremata: Tachyglossidae) from Australia. Eimeria tachyglossi has subspherical oocysts, 26.4 × 23.7 μm in size, with a single oocyst wall; no micropyle; four ellipsoidal sporocysts 13.2 × 9.7, slightly pointed at one end, each containing two sporozoites. Eimeria echidnae has subspherical oocysts, 19.4 × 17.8 in size, with a single oocyst wall; no micropyle; four ellipsoidal sporocysts 9.8 × 7.8, blunt at both ends, each containing two sporozoites. Octosporella hystrix has ovoid or subspherical oocysts 32.9 × 29.7 in size with a thick outer and thin inner oocyst wall; no micropyle; eight sporocysts spherical or slightly subspherical 11.3 × 11.2 each containing two sporozoites lying in embrace, with an extensive granular sporocyst residuum about the equator of the sporocyst. Endogenous stages considered to be of E. tachyglossi at least, were recognized in the lamina propria and epithelium on villi in the small intestine of three echidnas.     

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.     

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.     

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.     

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.