Morphological and histopathological studies of Thelandros chalcidae (Oxyuroidea: Pharyngodonidae) infecting Chalcides ocellatus from Egypt

Thelandros (Pharyngodonidae) is a gastrointestinal nematode parasite with a life cycle including lizards as main hosts. Thelandros chalcidae collected from the large intestine of the Egyptian ocellated skink, Chalcides ocellatus were described and illustrated by light and scanning electron microscopes. Seven out of fifteen (46.66%) of the examined lizards were found to be naturally infected. Also, host intestinal tissues were evaluated from hematoxylin/eosin-stained sections to describe any histopathological changes. Microscopic examinations revealed that the recovered pharyngodonid species characterized by mouth with triangular opening and surrounded by six simple lips, the cuticle had regular transverse annulations extending from the posterior margin of the lips to the end of the body. Male was cylindrical with distinct truncated posterior end and measured 1.59–1.86 (1.64 ± 0.10) long and 0.29–0.37 (0.32 ± 0.01) in maximum width at the level of mid-body. Female measured 1.72–2.43 (1.85 ± 0.2) long and 0.36–0.49 (0.42 ± 0.01) maximum width at the mid-body level, terminated posteriorly in a short, stout spike. Histological studies observed structural alterations represented by leukocytic infiltration, villi atrophy, and muscularis degeneration. These changes were indicative of inflammatory and degenerative reaction due to Thelandros chalcidae infection. The present morphological study revealed that the recovered pharyngodonid species was Thelandros chalcidae causing pathological alterations in Chalcides ocellatus intestinal tissues.


Open Access
The Journal of Basic and Applied Zoology Page 2 of 7 Fol and Mostafa The Journal of Basic and Applied Zoology (2021) 82:61 species is still under discussion and no pathological studies have been undertaken to assess the health impact on their hosts. Therefore, our aim was designed to study the morphology of Thelandros chalcidae and its impact on Chalcides ocellatus.

Worm collection
Fifteen Egyptian lizards, Chalcides ocellatus were collected from Abu Rawash region, Giza, Egypt from May to September 2020. Lizards were transported alive to the laboratory, they were euthanized with an overdose of sodium pentobarbital, subsequently necropsied and examined for helminths. The intestinal contents were examined for helminths under a stereomicroscope (OLYMPUS, SZ51). The collected worms were washed several times in saline solution then preserved in 70% ethanol.

Microscopic examination
The recovered nematodes were transferred to lactophenol for clearance, then examined and photographed using light microscope (LEICA DM 750) supplied with a LEICA ICC 50 HD Camera. For scanning electron microscopy, the worms were fixed in a solution of 3% glutaraldehyde, washed in 0.1 M sodium cacodylate buffer (pH 7.4), dehydrated through a graded ethanol series (50%, 60%, 70%, 80%, 90% and 100%), and then dried at 30°c for 30 min using critical point drier "LEICA, EM CPD300". After complete drying, nematodes were mounted on SEM stubs, coated with gold and examined with JEOL JSM-5200 SEM (Tokyo, Japan) at 25 kV as accelerating voltage. All measurements (mean ± SD, followed by a range in parentheses) are in millimeters (mm).

Histopathological examination
For histological studies, target organ (large intestine) of the Egyptian lizards, Chalcides ocellatus were carefully isolated and washed out in saline water then fixed in 10% buffered formalin. Tissues were dehydrated and processed for paraffin wax embedding. Sections were cut by a rotary microtome and stained with hematoxylin and eosin, then examined for any histopathological lesions and photographed using LEICA DM 750 microscope supplied with a LEICA ICC 50 HD Camera.

Results
The helminthological examination of 15 ocellated skink Chalcides ocellatus revealed natural infection by Thelandros chalcidae with a prevalence of (7/15) 46.66%. The mean intensity of infection was 1-3 parasite per host.

Generally
The body was robust cylindrical, whitish in colour, tapering at both ends with prominent cuticle striations from cephalic extremity and continuing to posterior end. Oral opening surrounded by 6 small lips. The esophagus was muscular, cylindrical ending by a welldeveloped bulb which leads to the intestine. Small lateral alae present. Prominent sexual dimorphism.

Histopathological findings
Sections of the intestinal tissue revealed cellular infiltration around the embedded worms which peripherally displaced the muscle fibers. Furthermore, Fig. 1 a-c, f- moderate fibrosis in connective tissue was evident around this area, and severe lymphocyte infiltration ( Fig. 2A, B), erosion of the muscularis layer, lesions associated with severe fibrosis and an inflammatory response reached serosa layer. Finally, the effects were varied and distributed among the four layers of the intestine leading to disintegration of muscularis and serosa layers due to burrowing of the parasite (Fig. 2B, C) as well as degeneration and atrophy of villi, infiltrated inflammatory cells diffused in the lamina propria, hyperplasia of the mucosal glands were observed (Fig. 2D).

Discussion
Oxyurids are parasitic nematodes infecting reptiles that constitute a very interesting group although their knowledge remains scarce (Aho, 1990;Bush, 1990;Martin & Roca, 2004;Sharpilo et al., 2001). Species of genus Thelandros Wedl (1861) infect many reptilian hosts (Bursey & Goldberg, 2005;Dung et al., 2009). Walton (1941) reviewed the geographical and host distribution of the genus Thelandros and pointed out that many of the known species for this genus were recorded from North Africa. Petter and Quentin (1976) considered Parapharyngodon a synonym of Thelandros. However, Thelandros sp. differentiated from Parapharyngodon sp. based on the posterior end morphology in both sexes (Bursey & Goldberg, 2005) Parapharyngodon sp. males do not have a conical-shaped genital area, or an accessory piece. In contrast, Thelandros sp. males have a prominent genital cone with papillae disposed outside this cone and caudal appendage inserted sub terminally on the body (Bursey & Goldberg, 1999;Bursey et al., 2013). Females of Parapharyngodon sp. generally have a conical shaped tail with a thick pointed end, like a spike. However, Thelandros sp. females have varied tail morphology (Bursey et al., 2013;Ramallo et al., 2016;Velarde-Aguilar et al., 2015). Bursey and Goldberg (2005) allocated 30 species to the genus Thelandros and stated the main generic features for Thelandros species based on the pattern of the caudal papillae, spicule length and morphology of the anterior cloacal lip in males, location of the vulva and morphology of the posterior end of the female. The current pharyngodonid species revealed main features of genus Thelandros represented by tapering at both ends with prominent cuticle striations from cephalic extremity and continuing to posterior end. Male has a conical-shaped genital area with papillae disposed outside this cone and distinct truncated posterior end with single spicule while female posterior extremity extending in short, stout spike.
Oxyurids live in the stomach or intestines of all classes of vertebrates; attach to the mucosa cause a wide range of damages including tissue degradation, hemorrhage, inflammation and mesenteric visceral adhesions (Botella & Esteban, 1995;Mohammed et al., 2017;Naem et al., 2006). The present histological studies revealed numerous pathological alterations in different layers of the large intestine of Chalcides ocellatus infected with T. chaicidae in agreement with previous observations by Schaftenaar et al. (2000) who reported pathological changes in lizard intestines due to nematodes infection. Also, in the present study, complete destruction of intestinal layers represented by erosion of the surface epithelium reaches the muscularis and serosa layers with severe leucocytes infiltration and lesions due to burrowing of the parasite, in consistent with previous reports by Dunn et al. (1983); Botella and Esteban (1995) and Meguid and Eure (1996) who reported that the parasites may occasionally change their site of attachment causing erosion of surface epithelium reaches as deep as the muscularis mucosa results in total loss at the region of attachment. Additionally, histological examination revealed atrophy in intestinal villi, this state may be due to the feeding of parasites on the villi, leading to its disappearance and the lack of surface area of absorption and thus low efficiency of absorption of nutrients and mineral salts (Dunn et al., 1983;Molnar et al., 1994).

Conclusions
Based on morphological and morphometric evidence, the current parasite resembles Thelandros chalcidiae, as well as sharing the same host species, site of infection, and locality record. As a result, we can mark it as Thelandros chalcidiae. Furthermore, for the first time in Egypt, we focused on the pathological effects of T. chalcidiae on Chalcides ocellatus..