Sporulation of Eimeria tenella (Coccidia) Oocysts ...

Proc. Helminthol. Soc. Wash. 51(2), 1984, pp. 320-325

Sporulation of Eimeria tenella (Coccidia) Oocysts Revealed by Scanning Electron Microscopy1 M. S. LONG AND R. G. STROUT Department of Animal Sciences, Kendall Hall, University of New Hampshire, Durham, New Hampshire 03824 ABSTRACT: Using scanning electron microscopy, we studied the process of sporulation of Eimeria tenella oocysts. By means of a procedure involving double sticky tape, we opened oocysts at hourly intervals until sporulation was complete. The cytoplasm of freshly passed oocysts consisted largely of amylopectin granules. Changes during sporulation consisted of contraction of the cytoplasmic mass from the oocyst wall, the appearance of a matrix embedding the amylopectin granules, formation of an outer surface over the cytoplasm, and cytokinesis that resulted in the formation of four sporoblasts. The sporoblasts became triangular, their edges thickened, and they then elongated into sporocysts within the oocyst. We did not see the formation of sporozoites.

Using light and interference microscopy, Canning and Anwar (1968) and Wagenbach and Burns (1969) presented detailed descriptions of the structural changes occurring in sporulating Eimeria tenella oocysts, but the process of sporulation has not been followed by scanning electron microscopy. In this study we examined the cytoplasmic changes occurring in sporulating E. tenella oocysts by fixing and processing oocysts at 1 -hr intervals for viewing and study with an AMR 1OOOA scanning electron microscope. Materials and Methods We performed two trials, one with strain LS-18 (Merck & Co.) and the other with Lilly-65 (Eli Lilly Co.). In both trials we obtained oocysts by scraping the ceca of infected White Leghorn chicks, approximately 4 wk of age, 7 days (LS-18) or 8 days (Lilly-65) postinfection. We homogenized the cecal cores in a Waring blender for 1 min, followed by pepsin digestion according to Rikimaru et al. (1961). We then brought the contents to a total volume of 2 liters with tap water adjusted to a pH of 7.5 and allowed the free oocysts to settle overnight at 4°C. After removing the supernatant fluid by aspiration, we centrifuged the oocysts at 200 g for 10 min and resuspended them in 100 ml of 0.5% K2Cr2O7. To enhance sporulation, we bubbled the solution with air at approximately 22°C until sporulation was complete (40-48 hr). Because the oocysts could begin sporulation immediately upon removal from the chicks, we withdrew 2-ml aliquots at hourly intervals from the start of pepsin digestion until sporulation was completed at 48 hr. We centrifuged aliquots at 200 g for 10 min, resuspended the sediments in double distilled water, centrifuged again at 200 g for 10 min, and resuspended the sediment in 3.0% glutaraldehyde in sample tubes at 4°C.

We held the oocysts in glutaraldehyde for a minimum of 35 min and a maximum of 3 days. After fixation each aliquot of oocysts was drawn into a 5-ml syringe through a 4-inch 14-gauge cannula that was then replaced with a 13-mm syringe filter containing a type HA 0.45-Mm-pore membrane (Millipore Corp.). Throughout the fixing process the oocysts remained on the filter membrane as the fluids were discharged through the syringe. To change fluid without altering the oocysts or rupturing the membrane from reverse flow, we removed the filter containing the oocysts, reaffixed the cannula, and aspirated the next fluid into the syringe. We expelled the fluid through the filter and oocysts again. Two 5-min rinses in double distilled water followed glutaraldehyde fixation. Postfixation was with 1.0% OsO4 for 10 min followed by three 5-min double distilled water rinses. We dehydrated the samples by suspension for 10 min in 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, and three 100% ethanol treatments. We excysted sporozoites according to Doran and Farr (1962) as just described. After dehydration, we removed the filter membranes containing the oocysts from the filters and critical-point dried them with a 10-min exchange period for the CO2. We mounted these membranes on pin-type mounts, oocyst side up and then took 2 cm 2 of double sticky tape (3M Co.), formed a loop, and holding the tape with forceps we gently touched it to the surface of the filter. The oocysts that adhered to the loop were fractured by compression between the loop and another 2-cm2 segment of double sticky tape mounted on a pintype stubmount. We peeled the top tape from the bottom one, inverted it, and mounted it next to the bottom tape segment. We sputter-coated all mounts with 200 A of gold-palladium for 4 min and then examined them in the scanning electron microscope. The filter membrane mounts provided a check on oocyst population and condition before fracturing. We took photographs with Polaroid type 55 positive-negative film.

Results From a senior thesis. Scientific Contribution Number 1212 from the New Hampshire Agricultural ExWe verified the results of Nyberg and Knapp periment Station. (1978a, b) that oocyst structure was not signifi320 1

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cantly affected by the glutaraldehyde within a period of several days. The wall of the freshly passed oocysts appeared smooth with no micropyle evident (Fig. 1). After sporulation, in some instances internal features were perceptible (though not clearly distinguishable) when higher voltages were applied (Fig. 17). The cytoplasm of freshly passed oocysts consisted largely of numerous granules, approximately 0.67 jum (±0.5 nm) long, identified by Wang et al. (1975) as amylopectin (Figs. 2, 3). Initial changes during sporulation consisted of a contraction or shrinkage of the cytoplasmic mass (Fig. 3) away from the oocyst wall, and the appearance of a matrix embedding some of the amylopectin granules (Fig. 4), although in some oocysts many internal granules remained separate and free from one another. We also noted short fiberlike structures throughout the granules (Fig. 4). As sporulation progressed, the outer surface of the cytoplasm formed a distinct but discontinuous cover that separated the cytoplasm clearly from its oocyst wall (Figs. 5,6). The first indication of cytokinesis was evident after approximately 20-22 hr. Cytokinesis was complete with the final separation into individual sporoblasts (Fig. 9) that soon appeared as rather uniform spheres (Fig. 10). The individual sporoblasts then became more flattened and triangular (Fig. 11) after approximately 24 hr of sporulation, followed by thickening of their outer edges (Fig. 12) and the formation of distinct membranes (Fig. 13). Many large pores were present at this stage of development (Figs. 11, 13). As the sporozoites developed, the sporoblasts thickened (Fig. 14) and elongated into sporocysts (Fig. 15) with smooth, firm walls (Fig. 16). The oocyst walls remained smooth within and without (Figs. 6-9, 11, 14), and were sufficiently thin and translucent that individual sporocysts (Fig. 17) could be seen under high voltage electron microscopy. We did not see stieda bodies clearly although we noted projections (Figs. 15, 17) that corresponded closely in location and shape with these structures. When we excysted intact sporulated oocysts (Fig. 17), typical sporozoites (Fig. 18) emerged. Discussion The stages of sporulation of Eimeria tenella described in this paper corresponded well with the description of sporulation in E. maxima by Canning and Anwar (1968) and with that of Wagenbach and Burns (1969) for E. tenella, ex-

cept that certain internal structures revealed by phase microscopy were not apparent by scanning electron microscopy. Scanning EM showed surface structures more readily than light microscopy and contributed an interesting and different view of the remarkable process of sporulation. We did not see distinct nuclei, chromosomes, or micropyles, nor did we observe the meiotic spindle reported by Canning and Anwar (1968) and Wagenbach and Burns (1969). Because we sporulated the oocysts at approximately 22°C, sporulation was more prolonged than that reported by Canning and Anwar (1968) and Wagenbach and Burns (1969). We noted a synchrony of events to some extent but not all oocysts sporulated at the same rate and assigning specific changes in development to specified hours of sporulation was difficult. The granules present in the early oocysts corresponded in shape and size to the amylopectin granules reported by Wang et al. (1975), which gradually diminished in number during sporulation and provided an energy source for the process. The nature and source of the embedding matrix (Fig. 3) was unclear, but it may have been the lipid or lipid complex that Wagenbach and Burns (1969) separated during high-speed centrifugation. The significance of the fibrous, rodlike structures (Fig. 4) was not clear and we could only speculate they might be associated with the amylopectin granules or perhaps be integrated into the envelope covering the sporoblasts and sporocysts yet to form. Canning and Anwar (1968) described filamentous chromosome material at approximately this time in sporulation, but the structures we saw were not enclosed within a nuclear membrane (Fig. 4). We saw no cytoplasmic vesicles as described by Wagenbach and Burns (1969) on the surface of the zygote, but during early cytokinesis we noted the appearance of porelike structures (Figs. 6, 7) that were soon obliterated by the membranes of the sporoblasts. Pores reappeared again in the triangular stage (Figs. 11, 13), but their significance was not apparent. Cytokinesis appeared first as an invagination of the cytoplasmic mass (Fig. 6), progressing rather rapidly (Figs. 7-9) to completion (Fig. 10). In late cytokinesis distinct overlying shroud or membrane covered the developing sporoblasts (Figs. 8-10) and may have been incorporated into their walls. One of the more confusing issues that arose


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Figures 1-6. Scanning electron micrographs of E. tenella undergoing sporulation. 1. Freshly passed intact oocyst before being opened. 0 hr, x 3,628. 2. Freshly passed oocyst, broken open, containing cytoplasm with amylopectin granules throughout. 0 hr, x 4,700. 3. Oocyst showing cytoplasm shrinking away from oocyst wall. 2 hr, x 5,260. 4. Oocyst showing amylopectin granules embedded in cytoplasmic matrix containing numerous fiberlike projections (arrows). 4 hr, x 11,000. 5. Oocyst showing progressive development of a distinct porous surface of the cytoplasmic mass, separating it from the wall of the oocyst. 8 hr, x 5,500. 6. Early cytokinesis showing the invagination of the cytoplasmic mass. The surface material is more dense with distinct openings. Note polar body (arrow). 12 hr, x 3,628. The time in hours of sporulation is approximate for all figures.


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Figures 7-12. Scanning electron micrographs of E. tenella undergoing sporulation. 7. Oocyst in intermediate cytokinesis. Note pores in cytoplasm. 13 hr, x 4,346. 8. Late cytokinesis with four sporoblasts forming. Note distinct overlying membrane (m). 14 hr, x 5,250. 9. Cytokinesis nearly complete into sporoblasts. Membrane is still evident. 20 hr, x 4,440. 10. Sporoblasts have separated completely in oocyst on left. Note membrane over sporoblasts in oocyst on right, perhaps incomplete cytokinesis. 22 hr, x 5,360.11. Triangular sporoblasts. Narrow end may become stieda body. Distinct membrane not apparent. 24 hr, x 3,900. 12. Triangular sporoblast with primordial sporozoites (arrow) forming from thickening periphery. 25 hr, x 3,430. The time in hours of sporulation is approximate for all figures.


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Figures 13-18. Scanning electron micrographs of E. tenella undergoing sporulation. 13. Sporoblast showing numerous pores. 26 hr, x 3,870. 14. Two of the four sporoblasts are beginning to thicken. Note inner wall of oocyst. 28 hr, x 3,430.15. Sporoblasts have elongated to sporocysts. End with stieda body (sb) can be recognized. Note polar body (arrow). 28 hr, x 2,681. 16. Three of four mature sporocysts, completely formed with smooth walls. 30-36 hr, x 4,151. 17. Three of four sporocysts, one with stieda body (sb), showing through the oocyst wall. 38 hr, x 3,604. 18. Excysted sporozoite. x 8,365. The time in hours of sporulation is approximate for all figures.


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during this study was the appearance and disappearance of a distinct membrane around the sporoblasts. We did not see membranes form but one must have been present throughout cytokinesis (Figs. 6, 7), not just at its completion (Figs. 8, 9). The distinct membrane apparent in Figures 8 and 9 disappeared at the triangular stage (Fig. 11) and was either replaced by or incorporated into another membrane for each specific sporoblast (Fig. 13), preceding the formation of the walls of the sporocysts (Figs. 14-16). The transition from the spherical sporoblasts to the flatter triangular form (Fig. 11) was rapid, followed by a pronounced thickening of its edges (Fig. 12), which apparently were destined to become the sporozoites. The more pointed end of the triangle perhaps will become the stieda body. Our methods did not reveal the process of sporozoite maturation except that the sporozoite primordia appeared to be the distinct peripheral folds seen in the late triangle stages. In two instances we saw what we interpreted to be polar bodies (Figs. 6, 15). Throughout sporulation the internal wall of the oocysts appeared smooth. As the sporoblasts matured to sporocysts, they elongated with a distinct anterior end appearing as the stieda body (Fig. 15). Viable motile sporozoites emerged (Fig. 18) following excystation in vitro.

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Acknowledgments

We gratefully acknowledge the technical help of Marilyn M. Ecker with the scanning electron microscope and the Oliver Hubbard fund for the financial support that made this study possible. Literature Cited Canning, E. U., and M. Anwar. 1968. Studies on meiotic division in coccidial and malarial parasites. J. Protozool. 15:290-298. Doran, D. J., and M. M. Farr. 1962. Excystation of the poultry coccidium, Eimeria acervulina. J. Protozool. 9:154-161. Nyberg, P. A., and S. E. Knapp. 1978a. Effect of sodium hypochlorite on the oocyst wall of Eimeria tenella as shown by electron microscopy. Proc. Helminthol. Soc. Wash. 37:32-36. , and . 1978b. Scanning electron microscopy of Eimeria tenella oocysts. Proc. Helminthol. Soc. Wash. 37:29-32. Rikimaru, M. T., F. T. Galysh, and R. F. Shumard. 1961. Some pharmacological aspects of a toxic substance from oocysts of the coccidium Eimeria tenella. J. Parasitol. 47:407-412. Wagenbach, G. E., and W. G. Burns. 1969. Structure and respiration of sporulating Eimeria stiedae and Eimeria tenella oocysts. J. Protozool. 16:257-263. Wang, C. C., R. M. Weppleman, and B. Lopez-Ramos. 1975. Isolation of amylopectin granules and identification of amylopectin phosphorylase in the oocysts of Eimeria tenella. J. Protozool. 22:560564.
