Coccidiosis of domestic rabbit (Oryctolagus cuniculus) in Egypt: light microscopic study
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- El-Shahawi, G.A., El-Fayomi, H.M. & Abdel-Haleem, H.M. Parasitol Res (2012) 110: 251. doi:10.1007/s00436-011-2479-0
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.
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.
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
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.
Characteristics of sporulated oocyst of species of Eimeria from rabbits mean (range)
Sporulation time at 25 ± 3°C (h)
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).
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.