DOI: 10.2478/s11686-009-0015-5 © 2009 W. Stefan´ski Institute of Parasitology, PAS Acta Parasitologica, 2009, 54(2), 85–89; ISSN 1230-2821 Endogenous stages of Choleoeimeria baltrocki (Daszak et Ball, 1991) n. comb. infecting the gall bladder of gold skink, Eumeces schneiderii Daudin, 1802 from Egypt

Abdel-Azeem S. Abdel-Baki1,2*, Huda M. El-Fayomi2, Thabet Sakran2 and Heba M. Abdel-Haleem2 1Zoology Department, College of Science, King Saud University, P.O. Box 12455, Riyadh 11451, Saudi Arabia; 2Zoology Department, Faculty of Science, Beni-Suef University, Egypt

Abstract Examination of 98 gold skink, Eumeces schneiderii Daudin, 1802 from Alexandria and Marsa-matroh Governorates, Egypt re- vealed oocysts of Choleoeimeria baltrocki (Daszak et Ball, 1991) n. comb. in the gall bladder and faeces. The prevalence of the infection was 40.8%. Sporulated oocysts were found in the faeces and the gall bladder contents. Sporulated oocysts were tetrasporocystic, cylindrical, reaching a mean of 38.7 (36–42) × 19.9 (17–25) µm with bilayered smooth and colourless wall. The dizoic sporocysts were subspherical and measuring 10.8 (9.5–13) × 9.3 (8–10.5) µm with a large globular sporocyst residuum. Sporocyst wall was bilayered joined by meridional suture. Sporozoites were crescent-shaped, blunt at one end and slightly tapered at the other. Endogenous stages (meronts, gamonts, gametes and young oocysts) were confined to the gall blad- der epithelium and the infected cells were hypertrophied. Based on the morphological features of the exogenous stages and en- dogenous development of the present parasite, its generic affiliation was revised and placed into the genus Choleoeimeria.

Keywords Choleoeimeria, Eumeces, gall bladder, Egypt

Introduction and endogenous stages of Eimeria baltrocki Daszak et Ball, 1991 and to revise its generic affiliation. Coccidia are among the most common parasites of reptiles worldwide. However, little is known about their biology and their diversity. Gold skinks Eumeces schneiderii Daudin, 1802 Materials and methods are widely distributed in Northern Africa and Middle East. Eimeria baltrocki Daszak et Ball, 1991 is the coccidian species During a survey of coccidian infection in lizards, 98 adult that known to parasitize E. schneiderii. The generic classifi- E. schneiderii (Reptilia, Scincidae) were collected from Alex- cation of eimeriid coccidia of reptilian hosts is a matter of long andria and Marsa-matroh Governorates in Egypt from 2005 term discussion, because they represent a diverse assemblage to 2007. Lizards were kept separately in plastic cages for sev- of species differing in the basic morphology of the exogenous eral hours to collect faeces, which was macerated and exam- stages (oocysts) as well as their endogenous development ined for oocysts. Fresh faeces were suspended in 2.5% (W/V) (Modrý and Jirků 2006). potassium dichromate solution. The oocysts were concentrated Paperna and Landsberg (1989) proposed the genus Cho- using Sheather’s sugar flotation technique. Heavily infected leoeimeria to comprise some Eimeria and Eimeria-like coc- lizards were anaesthetized with diethylethere, dissected and cidia infecting the biliary epithelium of reptiles. Species of the bile was collected by puncturing the gall bladder with a finely genus Choleoeimeria have elliptical oocysts (L/W ratio 1.6– pointed glass pipette. 2.2) with endogenous development confined to the gall blad- To determine the site of infection, tissue samples from the der epithelia and lack a Stieda body. Therefore, the current stomach, duodenum, small and large intestine, cloaca, heart, study aims to investigate the characteristics of the exogenous lung, liver, gall bladder and kidney were fixed in 10% buffered

*Corresponding author: [email protected] 86 Abdel-Azeem S. Abdel-Baki et al. formalin. Fixed tissues were processed for histological studies, Results sectioned and stained with haematoxylin and eosin (H & E). Different endogenous stages were investigated and pho- Description of oocysts (exogenous stages): The sporulated tographed using a Zeiss 100 microscope equipped for No- oocysts (n = 40) are cylindrical and often slightly curved (Fig. marski interference contrast microscopy. Sporulated oocysts, 1). They measure 38.7 ± 1.34 (36–42) × 19.9 ± 1.8 (17–25), sporocysts and different endogenous stages were measured. All with shape index of 1.96 ± 0.17 (1.7–2.3). Oocyst wall is measurements are in micrometers and data are presented as smooth and colourless, about 1.0 thick with no micropyle. The mean ± SD followed by the range in parentheses and the wall consists of two layers: an outer very fine membrane and shape index (ratio of length/width “L/W”). an inner thicker one (Fig. 1). No oocyst residuum, polar body

Figs 1–7. Nomarski interference contrast (NIC) photomicrographs of freshly collected sporulated oocysts of Choleoeimeria baltrocki n. comb. from the gall bladder of Eumeces schneiderii: 1. Sporulated oocyst surrounded with bilayered wall; outer layer (ol) and inner layer (il) and containing four sporocysts (s); scale bar = 10 µm. 2. Sporulated oocyst with sporocyst residual body (sr); scale bar = 10 µm. 3. Mag- nified part of sporulated oocyst focusing on the sporocyst suture (arrowheads); scale bar = 30 µm. 4 and 5. Fully sporulated oocysts with free sporozoites (sp); scale bars = 10 µm. 6 and 7. Photomicrographs showing non-infected and infected gall bladder epithelium with Choleoeime- ria baltrocki n. comb. 6. Normal epithelium in uninfected lizard; scale bar = 10 µm. 7. Low power view showing intense infection with hy- pertrophy of the infected cells towards the lumen (L); scale bar = 10 µm Choleoeimeria baltrocki n. comb. from E. schneiderii 87

or any other inclusions are observed. The four dizoic sporo- lium. No infection has been observed in the other examined or- cysts measure 10.8 ± 0.8 (9.5–13) × 9.3 ± 0.6 (8–10), with a gans. The infected gall bladders showed gross epithelial hy- shape index of 1.2 ± 0.11 (1–1.4). They are broadly ellipsoidal pertrophy and loosening of the subepithelial connective tissue with a smooth colourless unilayer sporocyst wall, which is (Fig. 7). composed of two valves joined by a longitudinal suture (Figs Histological sections revealed the presence of uninucleated 2 and 3). No Stieda body or any other thickening in the sporo- meronts which are oval and measure 10.5 ± 0.5 (9.5–11) × cyst wall is observed. A sporocyst residuum is present and 6 ± 0.4 (5.5–6.5) (Fig. 8). Multinucleated meronts are round consists of clusters of many granules. Sporozoites are cres- to oval, measure 16.3 ± 1.0 (15.5–17) × 13.8 ± 3.8 (11–16.5) cent shaped; enclosing the residuum (Fig. 4). With age, the and contain 12–14 merozoites (Figs 9, 10 and 11). Microga- sporocysts frequently dehisce and leave the sporozoites free in monts are round to oval, measure approximately 15.2 ± 2.8 the oocyst (Fig. 5). (13–20) × 14 ± 2.1 (11–17), and usually contain over 40 mi- Endogenous stages: Histological examination revealed a crogametes (Figs 8 and 11). Macrogamonts are ovoid to spher- high number of endogenous stages in the gall bladder epithe- ical, have a prominent nucleus (Figs 10 and 12) and measure

Figs 8–12. Photomicrographs showing various endogenous stages of Choleoeimeria baltrocki n. comb. infecting the gall bladder of Eume- ces schneiderii: 8. Uninucleated meront (M), microgamont (Mi) and zygotes or young oocysts (Zy); scale bar = 10 µm; 9, 10, 11 and 12. Macrogamonts (Ma), microgamonts (Mi), multinucleated meronts (M) and zygote (Zy); scale bars = 10 µm 88 Abdel-Azeem S. Abdel-Baki et al.

approximately 19 ± 3.2 (14–22) × 16.2 ± 2.7 (13–19). During ture. Three more assemblages can be traced within the latter maturation, the zygote or the young oocysts (Figs 8 and 11) are group: (1) species causing hypertrophy and displacement of elongated and later mature oocysts are released in large num- biliary epithelial cells, Choleoeimeria (Paperna and Landsberg bers in the bile secretion. 1989); (2) epicytoplasmatic species with endogenous stages in the gastrointestinal mucosa, Acroeimeria (Paperna and Lands- berg 1989) and (3) species with intracytoplasmatic develop- Discussion ment in the gastrointestinal mucosa. However, the majority of authors placed eimeriid coccidia from reptiles into collective Little is known about the biology and diversity of coccidian genus Eimeria (e.g. Daszak and Ball 1991, Upton et al. 1991, species infecting reptiles and amphibians. The number of coc- Telford 1997, Asmundsson et al. 2001 and Alyousif and Al- cidian parasites described from African reptiles is low in rela- Rasheid 2001). tion to the wide diversity of reptile fauna (Daszak and Ball Recently, molecular evidence indicates that Eimeria is pa- 2001 and Slapeta et al. 2003). Coccidia of the family Eimeri- raphyletic (Slapeta et al. 2001). Phylogenetic analysis based on idae parasitizing reptilian hosts comprise predominantly mem- nucleotide sequence of small subunit ribosomal RNA gene bers of the genera Caryospora Leger, 1904; Eimeria Schnei- confirmed separate status of the genus Choleoeimeria, which der, 1875 and Isospora Schneider, 1881 (Slapeta et al. 2003). probably forms a sister clade to the family Eimeriidae (Jirků There are five eimerian species with oocysts resemble to et al. 2002). those of the present study in both size and shape. Four of them Finally, Modrý and Jirků (2006) provided a revision of were described from the genus Eumeces and the other one was Eimeria-like coccidia infecting lizards of the family Scincidae described from genus Chalcides. These species are: E. sami and placed them into two genera, Choleoeimeria and Acro- Bovee, 1971 from Eumeces oshimensis; E. baltrocki Daszak et eimeria. They also listed some species, including the cur- Ball, 1991 from Eumeces schneiderii; E. fasciatus Upton et al., rently studied species Eimeria baltrocki, without sufficient 1991 from Eumeces fasciatus; E. egregia Telford, 1997 from data about their endogenous development and they stressed the Eumeces egregious and E. chalcides Probert et al.,1988 necessity of studying the endogenous stages of these species (Modrý and Jirků 2006) from Chalcides ocellatus. Oocysts of to allocate them to their correct genus and species. Eimeria sami, E. fasciatus, E. egregia and E. chalcides are Accordingly, the present biliary coccidium is classified as smaller in size than the oocysts of the present study. Moreover, a member of Choleoeimeria based on its oocysts morphology oocysts of E. sami are spherical shaped and oocysts of E. egre- and its endogenous development. gia are oval shaped while those of the present have long cylin- Oocysts of Choleoeimeria are typically shed in fully sporu- drical shaped oocysts. The morphology and measurements of lated form in the faeces of their hosts (Paperna and Landsberg E. baltrocki are similar to those of the present species which 1989, Lainson and Paperna 1999 and Lainson 2003) and are were also described from the same host. Considering the classified as having endogenous sporulation (Paperna and oocyst morphology and identical host, we consider found coc- Landsberg 1989). cidium to be conspecific with E. baltrocki. Daszak and Ball The oocysts of the present species were found mostly (1991) described E. baltrocki on the basis of the morphology sporulated in the faeces and gall bladder contents. This find- of oocysts collected from the faeces. The authors suspected that ing agreed with those mentioned by Sloboda and Modrý the gall bladder is the infection site which was confirmed in (2006). They assumed that sporulation is a time dependent this study. process and reported that oocysts remain in the gall bladder The correlation between oocysts’ shape index of more than content long enough to sporulate and are voided fully sporu- 1.4 and endogenous development in the gall bladder that was lated in the faeces. mentioned by Bovee and Telford (1965) was proved in the pres- Although, coccidian parasites are usually considered as ent study, since the shape index of the present parasite was 1.96. none or low pathogenic for their reptilian hosts (Levine 1988 However, many Eimeria species parasitizing reptiles have and Greiner 2003), the results of the current study showed been identified according to the morphology of their oocysts gross hypertrophy and loosening of the subepithelial connec- and sporocysts only. The current study focused on the eluci- tive tissue of the bladder. 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(Accepted January 7, 2009)