Morphological and Molecular Identication of Passalurus Ambiguus Rudolphi, 1819 in Domestic Rabbits (Oryctolagus Cuniculus) in Qena Governorate, Upper Egypt

Domestic rabbits in Egypt are used commercially for meat, but gastrointestinal disorders can affect production. Passalurus ambiguus is an intestinal parasite that infects the rabbit causing intestinal problems and death in severe cases. The present study collected domestic rabbits from several locations tgroughout the Qena Governorate in Upper Egypt. Passalurus ambiguus worms were detected in 90 out of 200 rabbits (45%). They were described morphologically using light and scanning electron microscopy. Males measured 4.622 mm (2.838–7.172 mm) in length and 0.278 mm (0.139–0.558 mm) in width. Females measured 5.622 mm (2.347–9.532 mm) in length, 0.314 mm, and (0.185–0.381 mm) in width. Phylogenetic results conrmed the identication of the worms as Passalurus ambiguus. They appeared as small white nodules in the appendix of the rabbits examined. Histopathologically, a heavy worm burden was observed inside the appendiceal lumen, among crypts, and inside the lymphoid follicles. The heavy worm infestation leads to hyperplasia in the epithelial lining of the appendix and the follicles resulting in lumen obstruction. Granulomatous reactions were induced due to irritation and injury by the worm. It could be concluded that morphological features, molecular phylogenetic data, and histopathological ndings clearly identied the present species as as Passalurus ambiguus Rudolphi, 1819. of this in order to the molecular phylogenetic links between Oxyuroid Enterobiinae, and Syphaciinae, with tribes in the subfamily Syphaciinae (i.e., Syphaciini, Acanthoxyurini, Higertiini, Passalurini, and Protozoophagini). The monophyly of Oxyurinae and Syphaciinae was conrmed by the current phylogenetic analyses. Thendings support the previous hypotheses (Hugot, 1988; Adamson, 1989; Hugot et al., 1996; Li et al., 2019; Cao et al., 2020). The current study is the rst to use phylogenetic analyses based on 28S sequence data to determine the systematic position of Passalurus ambiguus among Egyptian domestic rabbits. The Passalurus ambiguus clade was found to be monophyletic. This result was similar to Li et al. (2019) and Cao et al. (2020). Oxyuris equis formed a monophyletic group with Skrjabinema ovis and S. longicaudatum, consistent with the ndings of Li et al. (2019) and Cao et al (2020). data in GeneBank members


Introduction
The oxyurid Passalurus ambiguus can be found in the large intestine of both domestic and wild rabbits (Owen, 1972;Taffs, 1976). Moreover, it is the species with the best chances of adaptation to enteric culture farms (Grice and Prociv, 1993). Passalurus ambiguus infection of rabbits is not generally highly contagious. However, many pinworms are found in rabbits, and massive, fatal infections have been recorded in young rabbits (Owen, 1972). The parasite identi cation based on morphological characters is not fully reliable, leading to their misclassi cation. Therefore, it is important to expand the study area using PCR techniques to distinguish between different parasitic species.

A-Light microscopy
The gastrointestinal tract (GIT) was isolated and dissected, and the contents of the intestines were cleaned and sieved to eliminate the smallest particulates. As detailed by Georgi and Georgi (1990), nematodes were gathered using a stereomicroscope by screening diluted parts of intestinal material.
The recovered worms were xed in a mixture of 70% ethanol and 5% glycerine and subsequently mounted on a slide with drops of lactophenol then covered by a coverslip (Meyer and Olsen, 1975). Some worms were stained with acetic alum carmine, dehydrated in ascending grades of ethanol alcohol (70 %, 80 %, 90%, 95%, and 100%), then cleared in xylene and mounted in DPX. The detected worms were identi ed according to (Danheim and Ackert, 1929;Skinker, 1931;Hugot et al., 1983 Bellocq et al., 2001). PCR was carried out in a 25μl COSMO PCR RED Master Mix containing 1 μl template, 1 μM of each primer and Nuclease-free water to 50 μl. The following reaction conditions were used: (2 min initial denaturation at 95°C, then 25-35 cycles of 15 seconds at 95°C, 20 seconds at 50°C, and 30 seconds at 72°C. At 72°C, Post-PCR extension was performed for 1 minute). PCR products were examined on 2% agarose gels, stained with ethidium bromide, and photographed under a UV illuminator.
PCR ampli cation was puri ed using a DNA puri cation kit and subjected to automated DNA sequencing (ABI 3730XL DNA Sequencer, GATC Biotech, Germany) using the same primers used for PCR ampli cation.

B. Sequences of nematodes (partial 28S rDNA) from GenBank
The present study collected sequences from a partial 28S rDNA region for some related oxyuroid nematodes from GenBank. 13 partial 28S rDNA sequences from nematodes of the Superfamily Oxyuroidae (including outgroup from the Family Ascaridiidae) were obtained from the GenBank.
These sequences were compared to the sequence collected in this study (Table 1).

C. Analysis of DNA sequences
The 13 partial 28S rDNA sequences from nematodes were edited with GeneStudio TM Pro ssional Edition (version 2.2.0.0) and aligned with MEGA software (version 10.0.5) using default parameters.

D. Phylogenetic trees construction
On the basis of aligned partial 28S rDNA sequences, Oxyuroid nematode relationships were established using Maximum Likelihood (ML) and Maximum Parsimony (MP) approaches.
The ML and MP trees were obtained from the MEGA software (version 10.0.5). For ML analyses, the selected best-t model and parameters by Hasegawa-Kishino-Yano model with ve rate categories among sites (a discrete Gamma distribution [+G]) were used to construct an ML relationship tree. Bootstrap procedures were conducted with 1000 replications.

Histopathological analysis
Tissue specimens from the appendix were collected and xed in 10% buffered formalin. After proper xation, tissue blocks were embedded in para n. Thin (5 microns) sections were routinely prepared and stained with hematoxylin and eosin for the histopathological studies as described Intensity: 15-20 specimens of adult nematodes per infected rabbit.

A-Morphological analysis
The present study revealed the occurrence of Passalurus ambiguus that were seen by naked eye in the appendix and rectum of domestic rabbits ( g. 1 A). The enlarged appendix showing small white nodules ( g. 1 B). Worms attached in fecal pellets ( g. 1 C).

2-Scanning electron microscopy
In both males and females, transverse cuticular striations were visible on the body. Four papillae were found on the dorsal and ventral surfaces of P. ambiguus, indicating its anterior termination. The mouth was triangular and surrounded by three teeth (Figs. 7B, 8A, and 8B). No lips were seen.
Males of Passalurus ambiguus with a coiled posterior end, one short spicule (Figs. 7A, 7C, 7D, and 7E) protruding from the body in the cloacal region, and two pairs of large papillae around the cloaca, the second pair usually sessile (Figs. 7C, 7D, and 7E). In contrast, one couple was seen post cloacal as small and vestigial (Fig. 7D). The male body ends with a small papillary-like structure (Fig. 7E). The caudal appendix starts on its dorsal surface; however there is no pronounced striation.
Females of P. ambiguus have lateral wings in the anterior part of the body and plugs on the ventral surface. The tail was long, with noticeable loops, and it terminated with an exposed, pin-like tip (Figs. 8C and 8D). Prominent bands or annular structures (Figs. 8C and 8D) characterize the tail of mature females. As the worm approaches maturity, these bands increase in number and prominence, beginning at the posterior end of the large portion of the tail toward the anus. No bands could be detected in young or medium-sized specimens. The tail exhibits ne striations over the entire length, even showing through the annular bands.

B-Molecular analysis
Partial domains D1 and D2 of the 28S rDNA gene were ampli ed and sequenced for Passalurus ambiguus belonging to the families of Taeniidae Ludwig, 1886. The PCR ampli cation ranged from 700 to 800 bp. The sequence data of Passalurus ambiguus (781 nucleotides) examined were deposited in GenBank with accession numbers MZ571165.
The phylogenetic analysis was done using ML and MP methods. The obtained phylogenetic trees are shown in Figs. 9 and 10.
In the phylogenetic tree constructed by the ML method, representatives of the family Oxyuridae are distributed into two monophyletic clades. Clade 1 included Passalurus ambiguus, representing the subfamily Syphaciinae. Clade 2 included Oxyurisequis, Skrjabinemaovis, and S. longicaudatum, representing the subfamily Oxyurinae.
In the phylogenetic tree constructed by the MP method, Oxyurisequis separated from members of Skrjabinema, and it acts as a basal clade to the rest of included members of Oxyuroidea.
The present Passalurus ambiguus clustered together with the same species that having accession no. KY990018 with a strong bootstrap value (ML = 100, MP = 100). Skrjabinema ovis clustered together with Skrjabinema longicaudatum in a strong bootstrap value (ML = 100, MP = 100).
The genetic distance, estimated from 28S partial sequences, between the present Passalurus ambiguus and the previously recorded P. ambiguus was small (1.2%). The value between the Pharyngodonmicipsae, P. echinatus, and Thelandrosgalloti was small (1.7%). In contrast, high values between Thelandrosgalloti and T. tinerfensis and T. liformis were observed (10.6% to 10.8%), as shown in Table 3. Therefore, the genetic distances support is consistent with the constructed phylogenetic trees (Figs. 9 and 10).

C-Pathological ndings
The macroscopic appearance of the appendix was enlarged and lled with white nodules (Fig. 1A). The worms also appeared in the rectum pellets and were separated (Figs. 1B, C) The histopathological examination revealed numerous nematode worms (Passalurus ambiguous) infested the appendiceal layers, particularly inside the germinal layer of the lymphoid follicle and appendiceal lumen (Figs. 11, 12). The worm detection mainly inside crypts deeply into the follicles, causing hyperplasia in the lymphoid tissues and the follicular epithelium cells (Fig. 13). The transverse section of Passalurus ambiguous with the anterior and posterior portions contained eggs, displayed beneath the hyperplastic cells, where lymphocyte and eosinophil cells aggregated and surrounded it (Fig. 14). Granulomatous reaction was induced due to cells injury and aggregation of chronic in ammatory cells against the worm infestation, with in ammatory edema surrounded it (Fig. 15). Appendicitis manifested with a heavy worm infestation accumulated inside the lumen, leading to hyperplasia in the epithelial lining of crypts and follicles projected to form papillary formation causing narrowing and obstruction with worm and cell debris, besides hypertrophy of their follicles with reactive lymphocytes (Figs. 16 A, B). and the current study do not reference such a structure. It is possible that they mistook the head papillae, or the three teeth-like features, for lips, which led to the erroneous reference to such a morphological characteristic.

Discussion
In males, SEM gave a clear view of the cloacal region topography. There are three papillae pairs (the rst two pairs were pericloacal and larger than the last pair, which was small, sessile, and barely postcloacally located.). This nding is consistent with the description of a male P. ambiguus given by Hugot et al. (1983), Sultan et al. (2015) and Abdel-Gaber et al. (2019). Furthermore, another pair of small papillae has been observed, located in the region where the tail narrows and the caudal appendage begins, which is also a feature of male P. ambiguus described by Skinker (1931). It is well known that the number and position of papillae in the cloacal region are estimated differently between species. Skinker (1931) and Hugot et al. (1983) con rmed this variation based on the nature of the insemination process in Passalurus; researchers later used these features to distinguish P. ambiguus from other described species of Passalurus.
The tail topography of females distinguished P. ambiguus from P. nonanulatus, as the latter lacks the distinctive transverse cuticular striations that give P. ambiguus its moniliform appearance. Light microscopy can detect this unique appearance, but the SEM best observes it in accordance with those given by Sultan et al. There are three families of Oxyuroidea (Chabaud, 1974). The Pharyngodonidae includes parasites mainly found in the posterior gut of herbivorous lower vertebrates, with a few species parasitizing mammals (Petter and Quentin, 1976). The Oxyuridae and the Heteroxynematidae contain many species infesting mammals and only a few species infesting birds; these parasites are notably widespread in the caeca of lizards, terrestrial tortoises, marsupials, rodents, and primates.
The Oxyuridae are classi ed into three subfamilies, including Oxyurinae, Enterobiinae, and Syphaciinae, with tribes in the subfamily Syphaciinae (i.e., Syphaciini, Acanthoxyurini, Higertiini, Passalurini, and Protozoophagini). The monophyly of Oxyurinae and Syphaciinae was con rmed by the current phylogenetic analyses. The ndings support the previous hypotheses (Hugot, 1988 Mejia-Madrid (2018) observed that Skrjabinema ovis clustered with Passalurus ambiguus, and Oxyuris equis was a sister species to them in a monophyletic group with a signi cant support value (BI = 100). This may be explained due to insu cient data in the GeneBank for members of Passalurus and Skrjabinema.
The data set showed that Thelandros was paraphyletic, in which T. galloti was inserted within the Parapharyngodon clade. De Sousa et al. (2019) obtained this nding, but they reported that Thelandros seemed to be polyphyletic. According to Astasio-Arbiza et al. (1988), T. galloti shares some morphological characters associated with Parapharyngodon (i.e., caudal alae are absent and lateral alae are long and wide). Thelandros galloti, Parapharyngodonmicipsae, and P. echinatus were recorded from lacertids in Spain.
Our study revealed chronic in ammation due to worm irritation (Passalurus ambiguous) on the appendix layers, causing hyperplasia and hypertrophy in epithelial cells and lymphoid follicles with lumen obstruction. Some studies support the ndings of acute or chronic in ammation in pinworm-infested appendix specimens. However, most studies show that appendiceal pinworms cause fewer in ammatory changes (Panidis et al., 2011). Few studies have looked into the histopathology of Passalurus ambiguus in domestic rabbits (Mykhailiutenko et al., 2019). To our knowledge, this study is the rst focusing on the presence of P. ambiguus in the appendix of domestic rabbits with histopathological examination. In conclusion, the current ndings indicate that the pinworm species infecting Egyptian domestic rabbits is P. ambiguus Rudolphi, 1819.

Declarations Data availability
The materials used during the current study are available by the authors.

Ethics approval
The National Ethics Committee of South Valley University and veterinary authorities in South Valley University Province, Egypt, approved the method of this study.

Consent to participate
Not applicable.

Consent for publication
Not applicable.

Con ict of interest
The authors declare no competing interests.      In, intestine; Ov, ovaries; T, tail.    Maximum Likelihood (ML) tree based on the 28S sequence data showing the phylogenetic relationships of representatives of Oxyuroidea.
Ascaridia galli belonging to the family Ascaridiidae was selected as an outgroup. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches.   High power of the previous lesion (Fig. 13) to show the transverse section of Passalurus ambiguous (arrow) characterized with anterior and posterior portion containing egg inside its body, and surrounded with severely lymphocytes aggregation and eosinophils in addition to, hyperplasia in the epithelium lining (HY) of the crypts was noticed. (H&E., x40).