Parasitology Research

, Volume 110, Issue 1, pp 251–258

Coccidiosis of domestic rabbit (Oryctolagus cuniculus) in Egypt: light microscopic study

Authors

    • Zoology Department, Faculty of ScienceBeni-Suef University
  • H. M. El-Fayomi
    • Zoology Department, Faculty of ScienceBeni-Suef University
  • H. M. Abdel-Haleem
    • Zoology Department, Faculty of ScienceBeni-Suef University
Orignal Paper

DOI: 10.1007/s00436-011-2479-0

Cite this article as:
El-Shahawi, G.A., El-Fayomi, H.M. & Abdel-Haleem, H.M. Parasitol Res (2012) 110: 251. doi:10.1007/s00436-011-2479-0

Abstract

In the present study, the incidence and prevalence of coccidian infection among domestic rabbits in Egypt were investigated. Severe overall prevalence reaching 70% (70/100) was recorded. Eight species of Eimeria were detected. Mixed infection with three different species occurred most frequently. Eimeria intestinalis and Eimeria coecicola were generally the most predominant species. The complete life cycle of E. intestinalis was investigated. This study is the first to report coccidia in domestic rabbits in Egypt. Six species of Eimeria were reported for the first time.

Introduction

During recent years, rabbit industry became well established in Egypt (Lebdah and Shahn 2011). Most of the rabbit population in Egypt is in the hands of small holders, while the rest belongs to the commercial sector (Galal and Khalil 1994). Rabbit meat is used as a good source of animal protein, and some breeds are reared for fur production as well as for medical and biological purposes (Ragheb et al. 1999). Coccidiosis remains one of the most important infectious causes of digestive disorders in fattening rabbits (Vancraeynest et al. 2008). Rabbit coccidiosis is caused by parasites of the genus Eimeria, which are true pathogens that are always present in rabbit farms as they are virtually impossible to eradicate (Vancraeynest et al. 2008). Up till now, 15 Eimeria species are known to infect rabbits, and all of them are parasites of the intestinal tract, except for Eimeria stiedai, which invades exclusively the liver and the bilary tract (Li and Ooi 2009). The identification of these coccidia is based on the observations of the morphology of the oocysts, site of infection and clinical signs (Ceré et al. 1996). Despite the importance of rabbits as good source of animal protein in Egypt, very few parasitological studies of coccidian infection in rabbits have been done. To the best of our knowledge, the only available studies of Eimerian infection among rabbits in Egypt are those of Eid (1983), Abdel-Ghaffar et al. (1990), Arafa and Wanas (1996), Atta et al. (1999), Abdel Megeed et al. (2005) and Abu-Akkada et al. (2010). Nevertheless, most of these studies were restricted only to histopathology, biochemistry and treatment of the hepatic coccidiosis due to E. stiedai. The studies conducted by Eid (1983) are the only ones in Egypt on biology and life cycle of Eimeria magna infecting rabbits. Since the knowledge on rabbit coccidiosis is rather scarce, the present study was undertaken. The present study deals with the natural prevalence of Eimeria infections among rabbits and offers the description of both exogenous and endogenous stages of the most pathogenic Eimeria species found in Egypt.

Materials and methods

In the present study, 100 rabbits Oryctolagus cuniculus collected from Beni-Suef governorate, Egypt were investigated. Fresh faecal samples were examined for coccidian infection. Fresh oocysts were collected and concentrated by the usual floatation technique (Long et al. 1976). The morphometric data and the specific characteristics of unsporulated and sporulated oocysts were recorded.

Prepatent and patent periods

Six coccidian free rabbits (O. cuniculus) 3 months of age were inoculated with 1 × 105 oocysts of isolated pure strain of Eimeria intestinalis from the natural infection by single oocyst method (Pakandl et al. 2003; Li and Ooi 2009; Kvičerová et al. 2008). Two animals were left uninfected as controls. Animals were kept individually in the cages and fed on commercial food. The faecal pellets of each infected animal was collected daily and examined microscopically for the appearance and existence of the coccidian oocysts. The prepatent as well as patent periods were recorded.

Study of the endogenous stages

Eighteen coccidia-free rabbits were inoculated orally with approximately1 × 105 sporulated oocysts of the pure strain of E. intestinalis previously isolated. Always two of these animals were sacrificed at 24, 48, 72, 96, 120, 144, 168, 192 and 216 h post-infection (p.i.). Tissue samples were taken from the proximal and distal ileum and fixed in 10% neutral buffered formalin. Fixed tissues were processed for the usual histological studies for the light microscopy. Different endogenous stages were investigated, measured and photographed using photo research Olympus microscope equipped by a DP 25 digital camera. At least 30 specimens from each stage were measured. Four non-infected rabbits were kept under the same condition as control.

Results

During the present study, eight species of Eimeria, namely E. stiedai, Eimeria media, E. intestinalis, Eimeria coecicola, E. magna, Eimeria exigua, Eimeria perforans and Eimeria flavescens, were identified from naturally infected rabbits in Egypt. The overall prevalence was 70% (70/100). Mixed infection with three different species occurred most frequently. E. intestinalis and E. coecicola were generally the most predominant species, while E. magna, E. media and E. stiedai were less common and E. flavescence, E. exigua and E. perforans were relatively rare.

Morphology and morphometry of oocysts

E. stiedai (Lindemann 1865) Kisskalt and Hartmann 1907

The sporulated oocysts of E. stiedai were ellipsoid and measured 26.5 ± 1.9 (24–29) × 13.1 ± 1.4 (11–15) μm. Oocyst wall was smooth and light pink in colour and possessed a micropyle. It was composed of two layers; an outer very fine membrane and a thicker inner one (Fig. 1). There existed an oocyst residuum. The four dizoic sporocysts measured 9.7 ± 0.8 (9–11) × 5.6 ± 0.3 (5–6) μm. They were ellipsoid with smooth and showed a single-layered sporocyst wall. The sporulation time was 55 h at 25 ± 3°C.
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Fig. 1

Light micrographs of freshly shed sporulated oocysts of the eight species of Eimeria collected from naturally infecting domestic rabbits Oryctolagus cuniculus in Egypt. Scale bar = 10 μm. 1 Eimeria stiedai, 2 Eimeria media, 3 Eimeria intestinalis, 4 Eimeria coecicola, 5 Eimeria magna, 6 Eimeria exigua, 7 Eimeria perforans, 8 Eimeria flavescens

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Fig. 1

Light micrographs showing the four merogonic cycles of Eimeria intestinalis in the ileum of experimentally infecting rabbits. Scale bar = 10 μm. 9 Uninucleated schizont (Sch) of the first generation, 24 h p.i. 10 Multinucleated schizont (Sch) of the first generation, 48 h p.i. 11 Mature schizonts of the first generation with fully developed merozoites in the parasitophorous vacuole (PV), 72 h p.i. 12 Mature schizonts (Sch) of the second generation with merozoites (Me) in the parasitophorous vacuole (PV), 120 h p.i. 13 Mature schizonts of the third generation with mature merozoites in the parasitophorous vacuole (PV), 168 h p.i. 14 Fourth generation schizonts (Sch) at different stage of development with differentiated merozoites (Me) in the parasitophorous vacuole (PV), 192 h p.i.

E. media Kessel 1929

The sporulated oocysts of E. media appeared ovoid to ellipsoid and measured 22.3 ± 1.6 (19–24) × 12.1 ± 1.6 (10–15) μm. The oocyst wall was smooth, light pink in colour with micropyle. It was composed of two layers: an outer, very fine membrane and a thicker inner one (Fig. 2). There existed an oocyst residuum. The four dizoic sporocysts measured 8.2 ± 0.8 (7–9) × 4.5 ± 0.9 (4–6) μm. They were ellipsoid, with a smooth and a uni-layered sporocyst wall. The sporulation time was 33 h at 25 ± 3°C.

E. intestinalis Cheissin 1948

The sporulated oocysts of E. intestinalis were pyriform in shape and measured 20.3 ± 2.0 (18–23) × 13.5 ± 1.1 (12–15) μm. The oocyst wall was smooth, appeared greenish brown in colour and was provided with micropyle. It was composed of two layers: an outer, very fine membrane and a thicker inner one (Fig. 3). There existed an oocyst residuum. The four dizoic sporocysts measured 7.6 ± 1.1 (6–9) × 4.3 ± 0.4 (4–5) μm. They were ellipsoid and had a smooth and uni-layered sporocyst wall. The sporulation time was 60 h at 25 ± 3°C.

E. coecicola Cheissin 1947

The non-sporulated and sporulated oocysts of E. coecicola appeared cylindrical to elongate ellipsoid and measured 24.6 ± 2.5 (22–29) × 14.2 ± 1.5 (12–17) μm. The oocyst wall was smooth, yellowish green in colour and had a micropyle. It was composed of two layers: an outer, very fine membrane and a thicker inner one (Fig. 4). There was an oocyst residuum. The four dizoic sporocysts measured 8.6 ± 1.2 (7–10) × 5.4 ± 0.7 (5–6) μm. They were ellipsoid and possessed a smooth and uni-layered sporocyst wall. The sporulation time was 60 h at 25 ± 3°C.

E. magna Pérard 1925

The sporulated oocysts of E. magna were ovoid and measured 24 ± 1.3 (23–26) × 14.3 ± 1.0 (13–16) μm. The oocyst wall was smooth and appeared red brown in colour with micropyle. It was composed of two layers: an outer, very fine membrane and a thicker inner one (Fig. 5). There was an oocyst residuum. The four dizoic sporocysts measured 8 ± 1.4 (6–10) × 5 ± 0.8 (4–6) μm. They were ovoid and had a smooth and uni-layered sporocyst wall. The sporulation time was 45 h at 25 ± 3°C.

E. exigua Yakimoff 1934

The sporulated and non-sporulated oocysts of E. exigua were spherical in shape and measured 15 ± 0.8 (14–17) μm in diameter. The oocyst wall was smooth, purple in colour and did not show a micropyle. It was composed of two layers: an outer, very fine membrane and a thicker inner one (Fig. 6). There was no oocyst residuum. The four dizoic sporocysts measured 7 ± 0.9 (6–8) × 5.4 ± 0.5 (5–6). They were spherical and were covered by a smooth and uni-layered sporocyst wall. The sporulation time was 20 h at 25 ± 3°C.

E. perforans (Leuckart 1879) Sluiter and Swellengrebel 1912

The sporulated oocysts of E. perforans were ellipsoid and measured 15.6 ± 2.0 (12–18) × 10.3 ± 1.6 (8–11) μm. The oocyst wall was smooth, appeared greenish in colour and was provided with a micropyle. It was composed of two layers: an outer, very fine membrane and a thicker inner one (Fig. 7). There existed an oocyst residuum. The four dizoic sporocysts measured 5.3 ± 0.4 (5–6) × 4.5 ± 0.4 (4–5) μm. They were ellipsoid and were covered by a smooth and uni-layered sporocyst wall. The sporulation time was 25 h at 25 ± 3°C.

E. flavescens Marotel and Guilhon 1941

The sporulated oocysts of E. flavescens were ovoid, measured 23 ± 2.5 (22–30) × 16.2 ± 1.6 (14–18) μm. The oocyst wall was smooth, appeared brown in colour and did not show a micropyle. It was composed of two layers: an outer, very fine membrane and a thicker inner one (Fig. 8). There was no oocyst residuum. The four dizoic sporocysts measured 7.2 ± 0.6 (6–8) × 6.3 ± 0.4 (5–7) μm. They were ovoid and were covered by a smooth and uni-layered sporocyst wall. The sporulation time was 50 h at 25 ± 3°C.

Endogenous stages

Examination of the histological sections of the ileum revealed the presence of four merogonic cycles and each developmental stage was seen in a bright parasitophorous vacuole (Figs. 9–14). The early uninuclear schizonts of the first generation were observed at 24 h p.i. They were spherical in shape and measured 4 (3.5–5) × 3.5 (3–4.8) μm (Fig. 9). These schizonts grew in size reaching to 7 (6.4–8) × 6.5 (6–7) μm meanwhile their nuclei were divided repeatedly thus producing multinucleated schizonts at 48 h p.i. (Fig. 10). Mature schizonts of this generation were noted 72 h p.i. Each schizont contained 2–14 fully formed merozoites (Fig. 11). The measurements of the mature schizont were 14 (12–17) × 11 (10–14) μm. The first schizont cycle was completed in about 72 h post-inoculation, where fully formed merozoites were observed free within the parasitophorous vacuole (Fig. 11). Mature schizonts of the second asexual cycle were noted 120 h p.i. They measured 16 (13–18) × 11 (10–12) μm. Schizonts containing approximately 4–17 regularly distributed merozoites (Fig. 12). The developing schizonts of the third generation were detected 144 h p.i. They measured 13 (12–14) × 10 (9–11) μm. Mature schizonts of the third generation measured 18 (16–22) × 12 (11–14) μm were observed 168 h p.i. and they were estimated to produce 8 to 25 elongated merozoites (Fig. 13). The fourth generation schizonts appeared to be associated with the development of gamonts. Mature schizonts of the fourth cycle measured 19 (16–24) μm in diameter and contained approximately 8–32 merozoites (Fig. 14). These mature schizonts were detected 192 h p.i. The development of the sexual stages began about 144–192 h p.i., when merozoites of the third and fourth generations invaded the host cells and differentiated into micro- or macrogamonts (Figs. 15–20). The male determined merozoites of the third and fourth generations began to transform into microgamont as soon as they invaded the host epithelial cells of the ileum. The transformation process was observed to start with changing in shape to form ovoid to spherical early microgamonts. The early microgamonts of 15 (13–18) × 12 (11–14) μm were recognized by their large number of dark small nuclei distributed all over the whole surface of the gamont (Fig. 15). As the development of microgamonts proceeded, these nuclei migrated to be arranged at the periphery of the microgamont (Fig. 16). Later on, microgametes began to differentiate and numerous microgametes were noticed freely in the parasitophorous vacuole 168–192 h p.i. (Fig. 16). Young macrogamonts were firstly detected 144 h p.i. The macrogamonts were distinguishable from schizonts and microgamonts by their large central nucleus with a prominent nucleolus and the appearance of wall-forming bodies at their periphery (Figs. 17 and 18). The early macrogamonts were subspherical to spherical in shape (Fig. 17) and measured 12 (9–13) × 10 (8–11) μm. As the development proceeded, the nucleus became large and several dark homogeneous bodies known as wall-forming bodies appeared in the cytoplasm. At 168 h p.i., mature macrogametes of 17 (15–19) × 15 (14–17) μm were situated within a bright parasitophorous vacuole and contained many homogeneous dark wall-forming bodies being arranged at their periphery (Figs. 18 and 19). Numerous oocysts at different stages of maturation could be distinguished at 192 h p.i. (Figs. 19 and 20). Fully formed oocysts occurred in the faeces of the infected rabbits after 8 days p.i.
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Fig. 2

Light micrographs of microgametogenesis and macrogametogenesis of Eimeria intestinalis experimentally infecting rabbits. Scale bar = 10 μm. 15 Early microgamonts (Mi) with many nuclei. 16 Developing microgamonts (Mi) showing attached and detached microgametes (white arrows) in the parasitophorous vacuole (PV). 17 Developing macrogamonts (Ma) in the parasitophorous vacuole (PV). 18 Macrogamonts (Ma) in the parasitophorous vacuole (PV) with wall-forming body I (WFI), wall-forming body II (WFII) and nucleus (N). 19, 20 Developing oocysts (Oc) with oocyst wall (OW)

Discussion

During the present study, eight species of Eimeria were found to be present in the faeces of the investigated rabbits. According to the available literature, the study of rabbit Eimeria in Egypt is very limited. Most of these studies were concerned with histopathology, biochemistry, and treatment of the hepatic coccidiosis, and they were done mostly with E. stiedai (Abdel-Ghaffar et al. 1990; Arafa and Wanas 1996; Atta et al. 1999; Abdel Megeed et al. 2005; Abu-Akkada et al. 2010). Only one study was carried by Eid (1983) on the biology and life cycle of E. magna with electron microscopy. In the present study, six species were recognized and described for the first time in Egypt. Most species could be differentiated easily based on size, shape, colour, the presence or absence of an oocyst residuum and micropyle and sporulation time at 25 ± 3°C (Table 1). Our descriptions of the sporulated oocysts of Eimeria from rabbits in Egypt vary slightly in size and other minor characteristics from previous descriptions (Francalancia and Manfredini 1967; Cheissin 1968; Pellérdy 1974; Norton et al. 1979; Kasim and Al-Shawa 1987; Hobss and Twigg 1998; Razavi et al. 2010). Seventy percent of the investigated rabbits had mixed infections with at least three species. Kasim and Al-Shawa (1987) reported that 73% of rabbits were found to have mixed infection with three Eimeria species. Also, Toula and Ramadan (1998) reported five Eimeria species and found that 90% of the examined rabbits were positive and that mixed infections with two to three species were most frequent. Similarly, Yakhchali and Tehrani (2007) identified six species of Eimeria from rabbits and reported that mixed infections were common and 67% of the rabbit carried two to four different species. In another investigation, Gres et al. (2003) reported the same finding in wild rabbits (O. cuniculus).
Table 1

Characteristics of sporulated oocyst of species of Eimeria from rabbits mean (range)

Eimeria species

Oocyst

Sprocyst

Sporulation time at 25 ± 3°C (h)

Length

Width

Micropyle

Shape

Residium

Wall colour

Length

Width

E. stiedai

26.5 (24–29)

13.1 (11–15)

+

Ellipsoid

+

Light pink

9.7 (9–11)

5.6 (5–6)

55

E. media

22.3 (19–24)

12.1 (10–15)

+

Ellisoid

+

Yellow

8.2 (7–9)

4.5 (4–6)

33

E. intestinalis

20.3 (18–23)

16.5 (12–15)

+

Pyriform

+

Greenish brown

7.6 (6–9)

4.3 (4–5)

60

E. coecicola

24.6 (22–29)

14.2 (12–17)

+

Cylindrical

+

Yellowish green

8.6 (7–10)

5.4 (5–6)

60

E. magna

24 (23–26)

14.3 (13–16)

+

Ovoid

+

Red brown

8 (6–10)

5 (4–6)

45

E. exigua

15 (14–17)

15 (14–17)

Spherical

Purple

7 (6–8)

5.4 (5–6)

20

E. perforans

15.6 (12–18)

10.3 (8–11)

+

Ellipsoid

+

Greenish

5.3 (5–6)

4.5 (4–5)

25

E. flavescens

23 (22–30)

16.2 (14–18)

Ovoid

Brown

7.2 (6–8)

6.3 (5–7)

50

Measurements are based on50 oocysts of each species

Prepatent and patent periods

The prepatent period recorded for the E. intestinalis in the present study was 8 days, and this was identical in the study of Kvičerová et al. (2008). The minimum prepatent period recorded for other Eimerian species of rabbits was 4–5 days as in E. media, while the maximum prepatent period of other rabbit Eimerians was 16–18 days in E. stiedai (Anonymous 1977; Bhat et al. 1996). In the present study, the patent periods ranged from 6–7 days. Generally, in all rabbit intestinal Eimeria, the patent period lasts for 5–35 days, whereas in E. stiedai, it was 21–30 days (Bhat et al. 1996). Some studies found that maximum patent period reported for E. intestinalis was 10 days (Kvičerová et al. 2008).

Endogenous stages

In the present study, four asexual generations (schizogonic cycles) were observed in the epithelial cells of the small intestine. This did not agree with the earlier studies on E. intestinalis (Kheysin 1958; Pellérdy 1974; Peeters et al. 1984) in which only three schizogonic generations were reported. However, four asexual generations were reported for the same parasite in France (Licois et al. 1992). Generally, the exact number of asexual generations among the genus Eimeria is not fixed (Dai et al. 2005). During the present study, the first schizogonic cycle was completed in 72 h p.i. and this was similar to those reported by Licois et al. (1992) for the same Eimeria. However, Kheysin (1958) found the first generation at day 4 which was equivalent to the second generation seen in our study. Our fourth generation corresponded to Kheysin’s third generation but to the fourth cycle of Licois et al. (1992). The differences between our results and those of other studies are in timing of emergence and in the number of schizogonic generations. This may be due to the different doses of oocysts used at the beginning of infection (Licois et al. 1992; Pakandl et al. 2003). After a specific number of asexual generations, further merozoites are developed into gamonts rather than into further schizonts (Hammond 1973). In the present study, gamonts were developed mainly from the third generation merozoites where macro- and microgamonts were observed together with the fourth generation schizonts. These observations were in agreement with the results reported by Licois et al. (1992) in E. intestinalis and those of Jelínková et al. (2008) in E. exigua. Microgametogenesis described in the present study followed a similar pattern as in other coccidian parasites which occurred in two phases; growth phase and differentiation phase (Mehlhorn 2008). Different size of microgamonts and numbers of microgametes produced were reported for many species of Eimeria (Dai et al. 2005; Matsler and Chapman 2006; Mehlhorn 2006; 2008; Bashtar et al. 2010). In the present study, early macrogamonts were enclosed within a large parasitophorous vacuole and then contained a large central nucleus with a prominent nucleolus. These results coincided with those reported for many other Eimeria (Fayed et al. 1996; Al-Ghamdy et al. 2005; Bashtar et al. 2010). As the development proceeded, like other Eimeria, two types of wall-forming bodies were detected in the present study, large amounts of food reserve materials indicate the maturation of macrogametes (Dai et al. 2005; Mehlhorn 2006; Bashtar et al. 2010). After fertilization, the wall-forming bodies fused together giving rise to the oocyst wall. Oocysts with a double wall were shedded in the faeces of the infected host at the beginning 8 days p.i.

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