Zeitschrift f(Jr

Z Parasitenkd (1981) 66:1-7 Parasitenkunde Parasitology Research

�9 SpringerWerlag 1981

Original Investigations

Fine Structural Study of Eimeria truncata from the Domestic Goose (Anser anser dom.)*

R. Entzeroth 1, E. Scholtyseck 1, and I.Y. Sezen 2 1 Abteilung ftir Protozoologie, Zoologisches Institut der Universit/it, Poppelsdorfer Schlol3, D-5300 Bonn, Federal Republic of Germany 2 Institut ffir Anatomic, Physiologic und Hygiene der Haustiere (Forschungsstation fiir Geflfigelkrankheiten) der Universit/it, Katzenburgweg 7-9, D-5300 Bonn, Federal Republic of Germany

Abstract. Eimeria truncata oocysts were isolated f rom a naturally infected goose stock. Domest ic geese and ducks were inoculated with sporulated oocysts. Meronts , merozoites, macro- and microgamonts , and microgametes were studied with the light and electron microscope. These stages were located in the epithelial cells of the kidney tubules only. The meronts and merozoites showed the characteristic fine structures of Eimeria species. The macroga- monts only possessed one type of wall-forming bodies. It resembled the wall-forming bodies type I of other Eimeria species. One of the most promi- nent observations seemed to be the occurrence of osmiophil ic granular mate- rial in the paras i tophorous vacuoles o f all endogenous stages o f E. truncata.

Introduction

Eimeria truncata was first described by Railliet and Lucet in 1890. Its endogenous development occurs in the tubular epithelial cells of the kidneys of geese. Kotl /m (1933), Klimeg (1963), and Pel6rdy (1974) reported on the life cycle, the patholo- gy, host specificity, and the transmission of this kidney coccidium. The gaps in our knowledge of the life cycle o f E. truncata derive primarily f rom the failure to produce experimental infections, because in most experiments this parasite developed only in a few goslings. Until now no fine structural studies of E. truncata have been made. In this paper we report on experimental trans- missions and on electron microscope studies on E. truncata.

Materials and Methods

In the autumn of 1979 we discovered E. truncata oocysts in the kidneys of a 2-3 month old goose from a naturally infected stock. The oocysts were isolated by the trypsin method and stored in a 2.5% potassium dichromate solution at 4 ~ C. Experimental inoculations were performed using

* This paper is dedicated to em.o. Professor Dr. G. Piekarski on the anniversary of his 70th year

0044-3255/81/0066/0001/$01.40

Fig. 1. Developing merozoites (M) of Eimeria truncata in a parasi tophorous vacuole (PV) of an epithelial cell (HC) of the kidney tubules. A large residual body (RB) of the former meront is visible. The merozoites have normal structure with a conoid (C), rhoptries, a large mitochondrion, and in some cases a posterior polar ring (PP). Glutaraldehyde, OsO4, Vestopal W. x7,400

Fig. 2. Merozoites of Eimeria truncata in oblique and transverse sections within a parasi tophorous vacuole of an epithelial cell of the kidney of a gosling. Glutaraldehyde, OsO40 Vestapol W. x 7,600

Fig. 3. Developing merozoites of Eimeria truneata in a parasi tophorous vacuole (PV) of a host cell (HC). One merozoite with a posterior pole (PP) is pinching off from a large residual body (RB) of the former meront. • 7,900

Fine Structure of Eimeria truncata 3

26 goslings and 21 young ducks. Each animal was inoculated with 50,000 sporulated oocysts orally. The faeces of the inoculated birds were examined daily after the inoculation. The goslings used in this study were killed at 3, 5, 7, 9, 15, 21 days after inoculation and samples of the kidneys were removed and prepared for light and electron microscope studies. They were placed in 2.5% glutaraldehyde in cacodylate buffer for 6 h at 4 ~ C. After postfixing in 1.5% OsO4 for 4 h dehydration took place in 30% and 50% acetone. For poststaining 1% phosphotungstic acid was used. The tissues were embedded in Vestopal W. After polymerization the specimens were sectioned with glass knives on a Reichert ultramicrotome Ultracut. These sections were mounted on copper grids and stained with lead citrate prior to their examination with a Zeiss EM 9 $2 electron microscope.

Results

In the faeces of experimentally infected geese we found unsporula ted oocysts beginning on the 15th day postinfection (p.i.) These were allowed to sporulate which was completed within 3~4 days. The domestic ducks were not infected by E. truncata oocysts. Unsporula ted and sporulated oocysts were egg-shaped and had a prominent micropyle at the pointed pole. They measured 21.4 lain in length and 15.7 lain in width. Sporulated oocysts possessed four sporocysts with two sporozoites each (Sezen 1980). The developmental stages of E. truncata

(meronts, merozoites, macrogamonts , microgamonts) described using the elec- t ron microscope were located in the epithelial cells of the kidney tubules of geese 15-20 days p.i.

M e r o n ts

The first stages observed were meronts with fully developed merozoites (Figs. 1, 2, and 3). Some young merozoites were still a t tached at the residual body (RB) showing a posterior polar ring (PP). On ultrathin sections 20-25 merozoites were observed. Usually a large residual body was present. It conta ined mi tochon- dria, endoplasmic reticulum, and was bounded by a unit membrane. The meronts were lying in a well-developed paras i tophorous vacuole (PV) which contained amorphous osmiophilic material (GM). In most cases the host cell cytoplasm (HC) was reduced to a small band.

M e r o z o i t e s

The merozoites (M) had a length of 4 6 gm and a width of 2.5-3.5 ~tm. They showed the typical coccidian fine structure: a three-membranous pellicle (PE), a conoid (C), micronemes (MN), rhoptries (RH), a large mi tochondr ion (MI) with tubular structures inside, a well-developed endoplasmic ret iculum (ER), and a normal ly structured nucleus (N) with osmiophil ic plaques.

M a c r o g a m on ts

The young mac rogamon t s were ovoid in shape 7-9 ~tm long. Figure 4 shows a young m a c r o g a m o n t which is characterized by the presence of a large nucleus and a prominent nucleolus. This developing stage is limited by a unit membrane which is underlayed in some places by the former merozoi te ' s inner membrane

4 R. Entzeroth et al.

Fig. 4. Young macroga- mont of Eirneria truncata in a parasitophorous va- cuole (PV) of a host cell (HC). The parasitophor- ous vacuole contains nu- merous osmiophilic gran- ules (GM). Parts of this granular material lie also within membrane- bounded vesicles of the parasite (FV). The para- site stage has a large nu- cleus (N) and a nucleolus (NU), mitochondria (Mt), and a welI developed en- doplasmic reticulum (ER). Glutaraldehyde, OsO4, Vestopal W. x l 9,900

complex (IM). In the marginal region of the macrogamont numerous mitochon- dria (MI) were present. The endoplasmic reticulum (ER) was well developed. In this early stage wall-forming bodies were not yet present, however, large membrane-bounded vesicles containing dark granular material (GM) were ob- served distributed throughout the cytoplasm. These obviously represent food vacuoles (FV) because the parasitophorous vacuole (PV) was also filled with such granular material. Some of these food vacuoles contained only a few osmiophilic granules. Parts of the parasitophorous vacuole were very narrow and not filled with dark granules. It seems that this granular material in the parasitophorous vacuole may be considered as a characteristic of the E. truncata

parasitism. Older macrogamonts contained wall-forming bodies (WF) which were

bounded by a membrane and which tie in the cytoplasmic ground substance (Fig. 5). The cytoplasm was filled with numerous ribosome-like structures which were accumulated in some places. Besides a prominent nucleus (N) and a charac- teristic nucleolus, vesicles (DV) which were bounded by two or three membranes, a well-developed endoplasmic reticulum, mitochondria (MI), and food vacuoles (FV) were present. The macrogamont in Fig. 5 was found 21 days p.i. and only had one type of wall-forming bodies (WF). They were homogeneously

Fine Structure of Eimeria truncata 5

Fig. 5. Macrogamont of Eimeria truncata showing a large nucleus (N) with nucleolus (NU), wall- forming bodies (WF) of one type, mitochondria (M/), a well-developed en- doplasmic reticulum (ER), and large vesicles (D V) limited by two mem- branes. In the parasito- phorous vacuole (PV) are few osmiophilic granules (GM). Glutaraldehyde, Os04, Vestopal W. x 14,400

osmiophilic and similar to wall-forming bodies of type I of other E i m e r i a species. Wall-forming bodies of the second type were not observed in E i m e r i a t runca ta

macrogametes in this study. The parasi tophorous vacuole (PV) of this older stage contained only a few osmiophilic granules (GM).

M i c r o g a m o n t s

The microgamonts of E. t runca ta were subspherical in shape. They measured about 7 ~tm in diameter. In the microgamont of Fig. 6 there are sections of two nuclei. The characteristic structure of this stage are two centrioles (CE) between a nucleus and the unit membrane which limited this stage. The cyto- plasm contained numerous ribosomes, mitochondria (MI), numerous strands of a well-developed rough endoplasmic reticulum (ER), and food vacuoles (FV) filled with osmiophilic granular particles. Similar granules (GM) were also pres- ent in the parasi tophorous vacuole (PV). The microgamont is located at the apical pole of the epithelial cells directly in the vicinity of the lumen (L) of the tubules.

6 R. Entzeroth et al.

Fig. 6. Microgamont of Eimeria truncata. It shows two sections of two nuclei (N), food vacuoles (FV), mitochondria (M/), endo- plasmic reticulum (ER), and centrioles (CE). The parasitopborous vacuole (PV) is filled with granu- lar material (GM). GIu- taraldehyde, OsO4, Vesto- pal W. • 13,000

Discussion

The merozoites of E. truncata observed and described in this study had a length of 4 6 pm and a width of 2.5-3.5 pro. The macrogamonts and macroga- metes averaged in diameter 7-9 gin, the microgamonts about 7 gin. These mea- surements are very small compared with other Eimeria species (Pell6rdy 1974). The oocysts, however, and those on electron micrographs measured 21.4- 15.7 lain. Therefore it is very difficult to bring into accordance the measurements of the macrogametes with those of the oocysts. A possible explanation for these results is given by Klime~ (1963). According to this author oocysts of E. truncata occurring in the kidney were smaller than those found in the drop- pings.

One of the most interesting findings in this study was the presence of only one type of wall-forming body in the macrogametes of E. truncata. This type corresponds to the wall-forming bodies I of other Eimeria species (Scholtyseck et al. 1971). These bodies are usually homogeneously osmiophilic, limited by a membrane, and situated in the ground substance of the cytoplasm. The wall- forming bodies of the second type, however, very often are sponge-like and lie within vacuoles of the ER or the Golgi complex. In macrogametes of Toxo-

Fine Structure of Eimeria truncata 7

plasma gondii and Eimeria falciformis (Scholtyseck 1979) both types of wall- forming bodies are homogeneously osmiophilic. So far E. truncata is the only Eimeria species having only one type of wall-forming body. Shibalova and Morozova (1979) also reported only one type of WF in macrogametes of the goose coccidium Tyzzeria parvula.

In our opinion the osmiophilic granular material inside the parasitophorous vacuole of all endogenous stages of E. truncata seems to be characteristic of this species since such material has not been observed in other Eimeria species. In this kidney coccidium the granular material may serve as nutrition which is taken in by micropores into food vacuoles and digested. The origin of this granular material is not known. There are still many gaps in the life cycle, pathogenicity, fine structural knowledge, and experimental transmission of this coccidian species.

C Conoid MIH Host cell mitochondrion CE Centriole MN Micronemes DV Vacuole limited by two membranes N Nucleus ER Endoplasmic reticulum NU Nucleolus FV Food vacuole PE Pellicle GM Granular material PP Posterior polar ring HC Host cell PV Parasitophorous vacuole IM Inner membranous complex RB Residual body L Lumen RH Rhoptries M Merozoite WF WalI-forming bodies MI Mitochondrion

References

Klime~ B (1963) Coccidia of the domestic geese (Anser anser domesticus). Zentralbl Veterinaermed [B] 10:427-448

Kotl~m S (1933) Zur Kenntnis der Kokzidiose des Wassergeflfigels. Die Kokzidiose tier Hausgans. Zentralbl Bakteriol [Orig A] 129:11-21

Pell~rdy L (I974) Cocccidia and coccidiosis. Akad~miai Kiado, Budapest Railliet A, Lucet A (1890) Une nouvelle maladie parasitaire de l'oie domestique, determinbe par

des coecidies. CR Soc Biol (Paris) 42:293-294 Scholtyseck E (1979) Fine structure of parasitic protozoa. An atlas of micrographs, drawings

and diagrams. Springer Verlag. Berlin Heidelberg New York Scholtyseck E, Mehlhorn H, Hammond DM (1971) Fine structure of macrogametes and oocysts

of coccidia and related organisms. Z Parasitenkd 37 : 1-43 Sezen IYI Entzeroth R, Greuel E, Scholtyseck E (1980) Untersuchungen an dem Nierencoccid

Eimeria truncata aus der Hausgans (Anser anser domesticus). Berl Muench Tieraerztl Wochenschr 93 : 474-476

Shibalova TA, Morozova TI (1979) Vnutriiadernoe razvitie makrogamet kokzidii Tyzzeria parvula. Tsitologia 21 : 969-972

Received October 16, t980