Eimeria tenella: Further studies on the development of the oocyst

Experimental Parasitology 113 (2006) 174–178 www.elsevier.com/locate/yexpr

Eimeria tenella: Further studies on the development of the oocyst Zhou Kefu¤, Wang Yingying, Chen Mei, Wang Lihong, Huang Shuichun, Zhang Jun, Liu Renhai, Xu Hong The Key Laboratory of Education Ministry for Cell Biology and Tumor Cell Engineering, School of Life Science, Xiamen University, Xiamen 361005, China Received 21 September 2005; received in revised form 4 January 2006; accepted 5 January 2006 Available online 22 March 2006

Abstract Aim: The present study investigated the processes of macrogametogenesis and oocyst formation of Eimeria tenella (Xiamen strain), including the formation of wall-forming body1 (WFB1) and wall-forming body 2 (WFB2), the club-shape body and the origin of the residual body during the transformation from a macrogamete to an oocyst. Method: Transmission electron microscopy was used to follow ultrastructural changes of the organelles during parasite development. Frozen section techniques and special staining were used to determine the chemical composition of the club-shape body. Results: Electron lighter WFB1 appeared earlier than the electron denser WFB2 during the process of cyst wall formation. WFB2 appeared to play a key role in cyst wall formation, whereas WFB1 may have a limited role in the wall-forming process. When two last generation merozoites entered the same host cell simultaneously, one of them grew well, but the other one was developmentally retarded, and became a residual body. Our study indicates that the content of the club-shape body are lipoidal in nature, not amyolpectin as suggested previously, because they stained black by Sudan black-B. Conclusions: During of macrogametogenesis and oocyst formation of E. tenella (Xiamen strain), WFB2 plays a major role in cyst wall formation. The residual bodies come from the undeveloped macrogametes. The club-body is lipoid; and lipometabolism is important energy resource in E. tenella development. © 2006 Published by Elsevier Inc. Index Descriptors and Abbreviations: Eimeria tenella; Macrogamete; Oocyst; Ultrastructure; Cyst wall formation; Frozen section

1. Introduction Intestinal coccidiosis, caused by various species of Eimeria, is an economically important disease of poultry and livestock throughout the world. Eimeria tenella, Eimeria necatrix, Eimeria acervulina, Eimeria maxima, Eimeria branetti, Eimeria praecox, and Eimeria mitis all cause coccidiosis in chickens. According to a recent estimate (Zhang and Zeng, 2005), coccidiosis inXicts heavy economic losses, estimated to be 2 billion dollars a year worldwide. E. tenella is the most virulent species in chickens, often with bloody diarrhea and lower weight gain. Although the developmental process of the various Eimeria stages within chicken *

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intestine have been studied extensively (An et al., 2001a,b; Lee and Millard, 1972; Li et al., 1985; Madden and Vetterling, 1977; Mclaren, 1969; Mclaren and Paget, 1968; Peter and Long, 1982), the detailed ultrastructural processes of some organelle development, particularly the function of WFB1 and WFB2, remain controversial due to variations in morphology and physiology in diVerent strains of the same species. It has been suggested that both WFB1 and WFB2 play a role in cyst wall formation; and WFB1 was electron denser in TEM sections and appears earlier than WFB2 (Li et al., 1985; Mclaren and Paget, 1968). It has been proposed that the club-shape bodies are consisted of amyolpectin, and is dehydrated via phosphorylation of amylopectin, which produces energy necessary for oocyst maturation sporozoite production (Ryley, 1969), However, some other studies suggested that it was lipoidal

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Fifty-one-day-old chicks (The Avian Broiler) were bought from Xiamen China TAI Agriculture, and were fed with coccidian-free food. At 14 days, 40 healthy chicks were chosen for the following experiments.

for 4 h, washed three times at 4 °C in the same buVer for 30 min each, post-Wxed in buVered 1% solution (OsO4, ddH2O, 0.05 MPBS 1:1:2 ratio) at 4 °C for 8 h, and washed again three times as above. After dehydration in a graded acetone series for 25 min at each step, the samples were placed in 100% acetone two times for 30 min each, transferred to a solution containing acetone and resin (2:1 ratio) for 6 h, and placed in another acetone–resin solution (acetone:resin D 1:2) overnight. The tissues were immersed in incomplete resin for 2 days, and then in complete resin for 4 days before observation under a light microscope. Semi-thin sections were stained with methylene blue–azur II. Ultra-thin sections were prepared for TEM if the tissue samples were found to contain the diVerent parasite stages. Ultra-thin sections were double contrasted with uranyl acetate and lead citrate and observed in a XMUATC-YQ-006 TEM (Hitachi).

2.2. Parasites and experimental infection

3. Results

Eimeria tenella (Xiamen strain), originally isolated from a single oocyst Zhou et al. (2005, 2004), were propagated at the animal housing facility of the Life-Science Institute of Xiamen University. Sporulated oocysts were isolated in the 2.5% sodium hypochlorite, and washed three times with distilled water, and counted using a hemacytometer under a light microscope. Each of 14 days old chickens was inoculated with 20,000 sporulated oocysts of E. tenella, about 15–20 experimental chickens died during the experiment procession. At 135th, 141st, 147th, 153rd hours after being infection, 3–5 chickens were killed every time, and the tissue samples of experimental chicken containing various stages of the parasite were taken from the caecuem, and were cut to 1 mm3 using with a microtome.

3.1. The formation of macrogamete and Sudan black-B staining of the macrogamete

(Lee and Millard, 1972; Zhou et al., 2005; Zhou, 2004), but lacked good experimental evidence. Previous reports suggested that the residual bodies in the vacuole originated from the macrogametes. Our present study was undertaken to investigate the ultrastructure and developmental processes of various cell organelles, including the WFBs, the club-shape body, and the residual body, during Eimeria parasite development within chicken endothelial cells. 2. Material and methods 2.1. Experimental animals

2.3. Preparation of froze-section samples Intestinal tissues from 150 h post-infection were Wxed in 10% formalin for 12 h, washed thoroughly in running water, and placed at ¡30 °C. The frozen tissue samples were cut into 6–8 m sections, placed on a glass slide, Wxed with 70% alcohol for 2 min, dried at the room temperature, and stained with Sudan black solution (0.3 g Sudan black in 100 ml of 70% alcohol). The stained samples were heated in a microwave 4–6 s (medium power setting), washed with tap water for 2 min followed by a wash with double distilled water, treated with 70% alcohol for 2 min for separating color, washed again with distilled water, and dried at room temperature. The samples were then covered with a coverslip and sealed with glycerine gelatin. The sections were visualized under a microscope and photographed. 2.4. Preparation of TEM samples A small amount of the infected tissue containing the diVerent stages of E. tenella were Wxed in 2.5% glutaraldehyde buVered with 0.2 M PBS, pH 7.8, at room temperature

After the last generation merozoites entered the host cells, the merozoites become round in shape, and transform into macrogametocytes. During this period, the rhoptry, polar ring, conoid and microneme reduce in the merozoite, and disappear in succession (Fig. 1A). In the early stage of macrogametocyte development, the macrogamete appears to be within a vacuole. The WFB1 appeared earlier than WFB2 with a sponge-like texture (Fig. 1B). The electrondense wall-forming body (WFB2) appeared later (Fig. 1C). The periphery of WFB1 was rough, but the periphery of WFB2 was smooth (Fig. 2A). There are numerous clubshape bodies, stained black by OsO4, scattered in the cytoplasm (Fig. 2A), and also intravacuolar microtubules of diVerent shapes linking the plasma membrane of macrogamete to the vacuole (Fig. 2B). Meanwhile, in the vacuole, a residual body that is similar to macrogamete with smaller club-shape bodies and WFBs can be seen (Fig. 2C). Sudan black stained the macrogamete black indicating the presence of lipid, but the host cell failed to stain (Fig. 2D). 3.2. The formation of oocyst After fertilization, the macrogamete develops into a zygote. As the parasite diVerentiated, the WFB1 and WFB2 gradually moved toward the plasma membrane (Fig. 2A). The electron dense WFB2 began to discharge its contents (Fig. 3A) and took part in wall formation. Initially three layers were seen (Fig. 3A), which then extended to Wve layers (Figs. 3B and C), Finally, the inner four layers merged into one layer, denser and homogenous oocyst wall (Fig. 3D). Although the WFB1 also gathered near the membrane, the morphology, and texture of WFB1 did not change as oocyst wall formation progressed, and the number and density remained the same (Fig. 3D).

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A

C

Hn

Sch

WF1 WF1

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WF2 B hN

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NU WF1 N WF1

V Fig. 1. A merozoite within a host cell and swells with rhoptry, polar ring, conoid, and microneme having disappeared (A) bar 4 m; Electron light wallforming body WFB1 appears in the early macrogametocyte (135 h section) (B) bar 5 m; Electron dense wall-forming body (WFB2) appears later than WFB1 (147 h section), (C) bar 8 m.

A

B

WF2 WF1 TU WF1 CL CL WF1 C

D

MA R V Fig. 2. Both WFB1 and WFB2 move towards inner plasma membrane of zygote and there are numerous club-shape bodies, stained black by OsO4, scattered in the cytoplasm (A) bar 1 m; Antenna and bulb like tubes extended to the vacuole (B) bar 1.5 m; The residual bodies present in vacuole (C) bar 2 m; Macrogametocytes stained by Sudan black, suggesting lipoidal nature (D) bar 50 m.

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B

WF2

WF2 WF2 WF2

C

D

WF1

WF1 WF1

CL

WF1

Fig. 3. WFB2 discharges its contents as numerous small particles, possibly the key ingredients for oocyst wall formation. Three layers can be seen in each cyst wall (arrow head) (A) bar 1 m; Five layers of a oocyst wall (B) bar 1 m; The third layer (middle layer) gradually becomes thicker and sawtoothshaped: still Wve wall layers present (C) bar 0.3 m; The cyst wall grows thicker and more homogeneous. Only two layers of wall are seen the numbers of the denser WFB2 are reduced, but the number and density of WFB1 does not change (D) bar 1 m.

4. Discussion In general, only one merozoite enters one host cell, although occasionally more than one merozoite may be present in a single cell (Zhou, 2004). If two merozoites enter one host cell, only one of them will develop to mature gametocyte, and the other may continue to grow for some time, but will not mature. Previous investigators (Ferguson and Brich-Anderson, 1977; Pittlo and Ball, 1979; Speer, 1973) considered that the residual body was excreted substances from macrogametocyte. Our results suggest that it has a structure similar to a normally developed macrogamete (Fig. 2C). One possible interpretation would be that multiple parasites in one host cell would be over crowded. The host cell may not be able to provide suYcient nutrition for the development of two macrogametes simultaneously, leading to the production of a residual body. Some of the earlier reports regarded that the electron-denser WFB2 appeared earlier than the losser WFB1 (An et al., 2001a;Chobotar and Sholtyseck, 1982). Using monoclonal antibodies speciWc for the two types of wall-forming bodies, Mouafo et al. (2002) found that WFB1, but not the WFB2, and could be detected in macrogamonts at 137 h p.i. Other investigators believed that both of them appeared at the same time (Liu et al., 1993; Li et al., 1985). In our current study, we found that, in the early period of macrogametocyte development, the WFB1 appears Wrst with a sponge-like consistency (at 135 h section, Fig. 1B), followed by the electron denser WFB2 (at 147 h section, Fig. 1C). These diVerences may be due to variation among diVerent strains of E. tenella from diVerent geographical areas. The club-shape body of the macrogametecyte and macrogamete had been widely

considered to consist of amyolpectin (Lee and Millard, 1972; Lee and Long, 1972; Ryley, 1969), and the amyolpectin was dehydrated by the phosphorylase of amylopectin (Chobotar and Sholtyseck, 1982), which would provide energy, and the energy is necessary for the maturation of oocyst. Some authors have suggested that it is lipoidal (Li et al., 1985), but provided no experimental evidence. We used frozen sections and showed the macrogamete could stain using Sudan black stain (Fig. 2D) indicating the presence of lipid. We also showed that the club-shape bodies could be stained by OsO4 on TEM (Figs. 2A, C and 3C), in contrast to some previous observations (An et al., 2001a,b), where the club-shape bodies did not stain. These results suggest that the club-shape bodies are lipoid in nature, and may provide energy for the development of the oocyst. As for the function of WFB1 and WFB2, previous reports have suggested that both of the WFBs contribute to oocyst wall formation (An et al., 2001a; An et al., 2001b; Mouafo et al., 2002), according to Mouafo (2002), with WFB1 contributing to the inner wall of the oocyst, and WFB2 contributing to the outer wall. However, in our TEM observations, WFB2 is likely to play the major role in oocyst wall formation, and the WFB1 plays a lesser function in this process. The process of oocyst wall formation goes from one to Wve layers and Wnally becomes a well-distributed oocyst wall (Figs. 3A and D), whose thickness at this stage is nearly the same as the mature oocyst (Zhou et al., 2005, 2004). This suggests that cyst wall formation is completed at this stage; however, no obvious changes, including density, and quantities inside the cytoplasm, are observed for the WFB1 (Fig. 3D). According to Mouafo et al. (2000), WFB1 may facilitate disruption of the oocyst of E. tenella

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when ingested by a host. They found that WFB1 in E. tenella macrogamonts had undergone a proteolytic process and gave rise to three polypeptides during the formation of oocyst wall. They observed the suture structure inside the oocyst wall and thought that this facilitates the mechanical, rather than chemical disruption of the oocyst of E. tenella in the gizzard of the host. According to our results, we suggest that the major component of the oocyst wall of E. tenella originates from the WFB2, and the WFB1 may function in formation of the suture of oocyst, or other structures which would facilitate disruption of the oocysts. The oocyst wall is an important part of the oocyts that provides a protective barrier for survival. Understanding the mechanism of its formation may facilitate the development of measures to control coccidian infection. The function of WFB1, however, is still uncertain and requires further investigation, especially at the molecular level. Acknowledgments This work was supported by The Key Laboratory of Education Ministry for Cell Biology and Tumor Cell Engineering, School of Life Science, Xiamen University (No:2005108) and Xiamen University (2002, NO:Y07010). We also thank Professors Lin Yuguang, Hong Lingxian, and Tian Huiqiao of Life Science Institute of Xiamen University for advice and support; and Mr. Ni Ziming of Engineering and Detection Center of Xiamen University for help in preparation of TEM samples. References An, Jian, Wang, Ming, Kong, Fanyao, Yin, Peiyun, 2001a. Ultrastructral studies on the macrogametogenesis and oocyst forming of Eimeria tenella. Chinese J. Vet. Sci. 21, 559–561. An, Jian, Wang, Ming, Kong, Fanyao, Yin, Peiyun, 2001b. Ultrastructure of the gametogenesis of the Chick coccidiae (Eimeria tenella). Acta Zoologica Sinica 47, 431–435. Chobotar, B., Sholtyseck, E., 1982. In: The Biology of the Coccidian. Edward Arnold, London, pp. 104–108.

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