Introduction

The genus Eimeria Schneider, 1875 comprises homoxenous coccidians possessing four dizoic sporocysts within the oöcyst. There are now more than 1,300 described species of Eimeria (see Duszynski et al., 2000) but the majority of them have been described based only on oöcyst morphology. Paperna & Landsberg (1989) separated the tetrasporocystic, dizoic oöcysts of reptiles into the genera Eimeria, Choleoeimeria Paperna & Landsberg, 1989 and Acroeimeria Paperna & Landsberg, 1989 (Protozoa: Eimeriidae), based on the site of infection and the mode of development of the endogenous stages. Species of the genus Choleoeimeria have elliptical oöcysts (length/width ratio 1.6–2.2), with endogenous development confined to the gallbladder epithelia and also lack a Stieda body.

Phylogenetic analysis based on nucleotide sequences of the small subunit ribosomal RNA gene has confirmed the status of the genus Choleoeimeria (see Jirků et al., 2002), indicating that it forms a sister clade to the family Eimeriidae. The generic separation of Choleoeimeria has thus become widely accepted and many new species belonging to this genus have been described or redescribed (e.g. Lainson & Paperna, 1999; Lainson, 2003; Modrý & Jirků, 2006; Sloboda & Modrý, 2006; Paperna, 2007; Abdel-Baki et al., 2008, 2009; Al-Quraishy, 2011; McAllister 2012a, b). These authors stressed the necessity of studying the endogenous stages of the eimeriid coccidians in order to enable their allocation to their correct genus and species. According to Jirků et al. (2002), since it is impossible to properly classify the species that were originally described without notes on the site of infection and the presence or absence of sporocyst sutures and Stieda bodies, these species should be treated as species inquirendae or incertae sedis until more information is available. This has led Paperna (2007) to postulate that those eimerian species that have been reported in the literature as developing in the gallbladder epithelium should have their generic status amended. Following these suggestions, we here investigate the characteristics of the exogenous and endogenous stages for an eimeriid originally described as Eimeria zarudnyi Alyousif & Al-Shawa, 2003 from the gallbladder of the worm lizard Diplometopon zarudnyi. We provide a new combination and subsequently place the species in the genus Choleoeimeria.

Materials and methods

Thirty adult specimens of the worm lizard Diplometopon zarudnyi Nikolskii were collected during a parasitological survey in Saudi Arabia. Samples were captured by hand during June, 2012 from Al-Thumamah (24°41′N; 46°42′E) in Riyadh, in the central region of Saudi Arabia. The lizards were kept separately in plastic cages for several hours in order to collect faeces which were then macerated and examined for oöcysts. Fresh faeces were suspended in 2.5% (W/V) potassium dichromate solution. The oöcysts were concentrated using Sheather’s sugar flotation technique. Heavily infected lizards were anaesthetized with diethyl ether, dissected, and bile was collected by puncturing the gallbladder with a finely pointed glass pipette. Selected tissues (stomach, intestine, liver, kidneys and muscles) were fixed in 10% buffered formalin. Fixed tissues were processed for histology using standard methods and paraffin sections were stained with haematoxylin and eosin. All stages were observed and photographed using an Olympus BX51 microscope with an Olympus DP71 camera according to the guidelines of Duszynski & Wilber (1997). Fifty sporulated oöcysts, and 10–20 endogenous stages were measured. All measurements are reported in micrometres; data are presented as the range followed by the mean ± standard deviation (SD) (where possible) in parentheses.

Results

Coprological examination revealed the presence of oöcysts of Choleoeimeria in five (17%) of the 30 lizards examined. This species is redescribed below.

Choleoeimeria zarudnyi (Alyousif & Al-Shawa, 2003) n. comb

Host: Diplometopon zarudnyi Nikolskii (Amphisbaenia: Trogonophidae) (type-host)

Locality: Al-Thumamah (24°41′N; 46°42′E), in Riyadh, in the central region of Saudi Arabia (type-locality).

Prevalence: 17% (5 out of 30 lizards).

Site of infection: Gallbladder.

Sporulation: Sporulation endogenous; both sporulated and unsporulated oöcysts were found in the gallbladder lumen and intestinal contents prior to being voided in the faeces.

Type-material: Photosyntype for the sporulated oöcysts and one slide with syntypes of haematoxylin-eosin stained endogenous stages in the gallbladder epithelia have been deposited in the parasitological collection of the Hungarian Natural History Museum under the inventory number HNHM-70392.

Description (Figs. 1–11)

Figs. 1–10
figure 1

Choleoeimeria zarudnyi (Alyousif & Al-Shawa, 2003) n. comb. from the gallbladder of the worm lizard Diplometopon zarudnyi. 1–4. Photomicrographs of freshly collected mature oöcysts with bi-layered wall (OL, outer layer; IL, inner layer), containing four sporocysts (S) with sporocyst residuum (SR) and two sporozoites (Sp). 5–10. Endogenous stages. Infected epithelium becomes hypertrophied and displaced to the lumen of the gallbladder being connected with the basal membrane by a thin pedicle (arrowheads). 5. Mature meront (M) with mature merozoites (Me). 6, 7. Microgamonts (Mi). 8–10. Macrogamonts (Ma) with wall-forming bodies arranged at the periphery (arrows) and a centrally located nucleus (N). Scale-bars: 10 μm

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Fig. 11
figure 2

Schematic drawing of a mature oöcyst of Choleoeimeria zarudnyi (Alyousif & Al-Shawa, 2003) n. comb. The oöcyst is with bi-layered wall (OL outer layer, IL inner layer) and contains four sporocysts (S), each with sporocyst residuum (SR) and two sporozoites (Sp) each with a refractile body (RB). Scale-bar: 10 μm

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Exogenous stages

Oöcyst without micropyle, elongate ovoid to ellipsoidal, rounded at ends, measuring 25–32 × 18–25 (27 × 22), with length/width ratio 1.0–1.3 (1.2) (Figs. 1–4, 11). Oöcyst surface smooth, wall bi-layered; oöcyst residuum and polar granule absent (Figs. 1–4). Four oval sporocysts present, with thick single-layered wall and without Stieda body (Figs. 1–4). Dimensions of sporocysts 10–13 × 6–9 (11 × 7), with length/width ratio 1.3–1.6 (1.5). Sporocyst residuum composed of a large number of granules differing in size (Fig. 3). Sporozoites within sporocysts usually laying head to tail, with two refractile bodies (Fig. 11). Sporozoites banana-shaped, measuring on average 13 × 3 (from ruptured sporocysts, see Fig. 4).

Endogenous development

Mature meronts spherical, measuring 12–15 (14 ± 0.6) in diameter, and estimated to produce 20–25 merozoites (Fig. 5). Microgamonts irregular in shape, measuring 9–12 × 7–10 (11 ± 0.4 × 8 ± 0.5) (Figs. 6, 7). Macrogamonts mostly subspherical (Figs. 8–10) with a prominent nucleus in the centre and wall-forming bodies at the periphery (Fig. 10), measuring 12–18 × 7–12 (14 ± 0.7 × 10 ± 0.8).

The intrinsic development occurred within epithelial cells lining the gallbladder and bile-duct. No endogenous stages were encountered in any other organ. The infected host cells were hypertrophied and protruded from the epithelial layer into the lumen (Figs. 5–10). Usually the infected cells maintained connection with the basal membrane only by means of a thin pedicle (Figs. 6, 7, 9).

Remarks

To our knowledge, three species of Eimeria and one species of Choleoeimeria have been described to date from among amphisbaenian lizards (Table 1). These are Eimeria amphisbaeniarum Huntington, Cisper, Smith, Powell, Parmerlee & Lathrop, 1996, E. witchery Huntington, Cisper, Smith, Powell, Parmerlee & Lathrop, 1996 associated with Amphisbaena manni Barbour (see Huntington et al., 1996), E. zarudnyi Alyousif & Al-Shawa, 2003 associated with D. zarudnyi (see Alyousif & Al-Shawa, 2003) and Choleoeimeria amphisbaenae Lainson, 2003 associated with Amphisbaena alba Linnaeus (see Lainson, 2003). Eimeria amphisbaeniarum and E. witchery have thinner oöcysts and a larger length/width ratio than those observed here, whereas the oöcysts of C. amphisbaenae are significantly longer and also have a larger length/width ratio. The morphology and measurements in the original description of E. zarudnyi are similar to those observed in the present material which is also described from the same host. Based on oöcyst morphology and identical host, we consider it conspecific with E. zarudnyi. Alyousif & Al-Shawa (2003) described E. zarudnyi on the basis of the morphology of oöcysts collected from the faeces. They suspected that the gallbladder was the site of infection and this was confirmed in the present study. Accordingly, the present biliary coccidium is classified as a member of the genus Choleoeimeria based on its oöcyst morphology and endogenous development.

Table 1 Comparative measurements (in μm) of Choleoeimeria zarudnyi n. comb. and morphologically similar species from amphisbaenian lizards
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Discussion

The descriptions of many species of Eimeria from reptiles are based solely on the morphology of their mature oöcysts and sporocysts following Levine (1973), with no consideration for any differences that might exist in the rest of the parasite life-cycles (Lainson, 2003). In general, oöcyst morphology can be quite diverse, both between and within host species, with the only constant feature being the presence of four sporocysts, each with two sporozoites (Asmundsson et al., 2001). It is impossible to be sure, therefore, of the generic status of many parasites without reference to the endogenous stages (Lainson & Paperna, 1999). Paperna & Landsberg (1989) proposed that the genus Choleoeimeria comprised some Eimeria and Eimeria-like coccidians infecting the biliary epithelium of reptiles.

Species of the genus Choleoeimeria have elliptical oöcysts (length/width ratio 1.6–2.2) with endogenous development confined to the gallbladder epithelia and lack a Stieda body. This separate generic status of the eimerians inhabiting gallbladders of reptilian hosts was not accepted until their distinctiveness from Eimeria spp. had been demonstrated through molecular phylogenetic studies of Jirků et al. (2002). Subsequently, many authors (e.g. Lainson & Paperna, 1999; Lainson, 2003; Modrý & Jirků, 2006; Sloboda & Modrý, 2006; Paperna, 2007; Abdel-Baki et al., 2008, 2009; Al-Quraishy, 2011, McAllister 2012a, b) have stressed the necessity of studying the endogenous stages of these species in order to allocate them to their correct genus and species. Paperna (2007) also suggested that those eimerian species that have been reported in the literature as developing in the gallbladder epithelium should have their generic status amended. The current study, therefore, investigated the characteristics of the exogenous and endogenous stages of E. zarudnyi Alyousif & Al-Shawa, 2003 and revised its generic affiliation to the genus Choleoeimeria.