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

Abstract

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 confirmed the identification 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 findings clearly identified the present species as as Passalurus ambiguus Rudolphi, 1819.

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 identification based on morphological characters is not fully reliable, leading to their misclassification. Therefore, it is important to expand the study area using PCR techniques to distinguish between different parasitic species.

Molecular methods have lately been employed to identify morphologically similar taxa, and they have greatly aided taxonomic, phylogenetic, and epidemiological research on several organisms (Yang et al., 2013; Ogedengbe et al., 2014; Li et al., 2015). These methods also provide excellent genetic markers for genetic variation in oxyurids, especially Passalurus ambiguus (Sheng et al., 2015; Abdel-Gaber et al., 2019; Solórzano-Garca et al., 2020). The goal of this work is to characterize the morphology of Passalurus ambiguus, which infects domestic rabbits in the Qena Governorate of Upper Egypt, in order to corroborate the molecular phylogenetic links between this species and other Oxyuroid species. Furthermore, the histolopathological consequences of this species in the appendix of the rabbits studied were the focus of this research.

Materials And Methods

Sample collection

Ethics approval. The institutional ethics committee. Animal experiments followed the institutional ethical guidelines for the care and use of animals in research to the letter. Approval for animal studies were obtained from the Faculty of Veterinary medicine, South Valley University, Qena, Egypt (Approval number:25/13/11.2021).

200 domestic rabbits (Oryctolagus cuniculus) were collected at various locations and markets in Qena Governorate, Upper Egypt, from October 2018 to October 2020. Rabbits were transported to the Parasitology Laboratory, Department of Zoology, Faculty of Science, South Valley University, Qena.

Morphological analysis

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 fixed 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 identified according to (Danheim and Ackert, 1929; Skinker, 1931; Hugot et al., 1983; Petter and Quentin, 2009; Sultan et al., 2015; Abdel-Gaber et al., 2019; Mykhailiutenko et al., 2019).

B- Scanning electron microscopy

Specimens were submerged in a solution of 3 percent gluteraldehyde buffered with 0.1 M phosphate buffer (pH 4) for 2-4 hours at room temperature. Tissues were washed three times in 0.1 M phosphate buffer (pH 7.2) for ten minutes each time. Specimens were postfixed in a light container for 2-4 hours at room temperature with 1-2 percent osmium tetroxide in 0.1 M phosphate buffer (pH 7.2). In graded ethanol/acetone solutions, dehydration occurs. Dehydration was done twice with 100% ethanol or acetone (15-30 minutes for each) (Hussein et al. 2018). Finally, worms were examined in the Central Laboratory of South Valley University using Jeol JSM-5500 LV Scanning electron microscope (Jeol, Japan) with a 20 KV accelerating voltage.

Molecular analysis

A. DNA extraction and sequencing

Specimens were removed from ethanol then distilled water was added to samples to completely remove the excess ethanol. The samples were placed individually in separate 1.5 ml microcentrifuge tubes, and DNA was extracted using genomic DNA mini kit (Quick- DNATM Funga/Bacterial Miniprep Kit).

PCR primers C1′: ACCCGCTGAATTTAAGCAT and D2: TCCGTGTTTCAAGACGG were used to amplify the D1 and D2 domains of 28S rDNA from whole DNA (de 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 amplification was purified using a DNA purification kit and subjected to automated DNA sequencing (ABI 3730XL DNA Sequencer, GATC Biotech, Germany) using the same primers used for PCR amplification.

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 Profissional 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-fit model and parameters by Hasegawa-Kishino-Yano model with five 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 fixed in 10% buffered formalin. After proper fixation, tissue blocks were embedded in paraffin. Thin (5 microns) sections were routinely prepared and stained with hematoxylin and eosin for the histopathological studies as described by (El-Akabawy et al., 2004).

Results

Taxonomic summary

Species: Passalurus ambiguus Rudolphi, 1819 (F: Oxyuridae)

Host: Domestic rabbits Oryctolagus cuniculus (F: Leporidae).

Locality: Qena Governorate, Upper Egypt.

Site of infection: Large intestine.

Prevalence: 45% (90 out of 200).

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 (fig. 1 A). The enlarged appendix showing small white nodules (fig. 1 B). Worms attached in fecal pellets (fig. 1 C).

1- Light microscopy

Male (Based on 12adult worms)

The length of the body is 4.622 mm (2.838–7.172 mm), the width is 0.278 mm (0.139–0.558 mm). The club-shaped esophagus measures 0.710 mm (0.391–0.1238 mm) in length (Figs. 2A, 3A, and 3C), the corpus measures 0.538 mm (0.274–0.908 mm) in length (Figs. 2B, 2C, 3B, 6A, and 6B), and the subspherical bulb measures 0.165 mm (0.094–0.304 mm) in diameter (Figs. 2B, 3B, and 6A). Testis (3A, 3C, and 6A) opens into thin-walled vas deferens. Vas deferens opens into the cloaca containing a single short protruded spicule measuring 0.094 mm (0.068–0.153 mm) in length (Figs. 2A, 2D, 2E, 3A, 3D, 6A, and 6C).

The cloaca was shown anteriorly by two pairs of large papillae (Figs. 2D, 2E, and 6C) and posteriorly by one pair of small papillae (Figs. 2D, 2E, 3D, and 6C). Two caudal papillae (Figs. 2D, 3C, and 6C) are present in the region of the tail. The body ended with a small coiled tail and measured 0.298 mm (0.145–0.492 mm) long (Figs. 2A, 2D, 3A, 3C, 6A, and 6C, and table 2).

Female (Based on 12 adult worms)

The body length is 5.622 mm (2.347–9.532 mm), and the width is 0.314 mm (0.185–0.381 mm). The club-shaped esophagus measures 0.484 mm (0.435–0.571 mm) in length (Figs. 4A and 5A), the corpus measures 0.369 mm (0.325–0.451 mm) in length (Figs. 4B, 4C, 5B, 6B, and 6D), and the subspherical bulb measures 0.124 mm (0.100–0.146 mm) in diameter (Figs. 4C, 5B, and 6D). Two large thin-walled ovaries (Figs. 5A and 6D) lie near the body wall and open posteriorly into the much-convoluted oviducts (Fig. 6D) packed with eggs in mature females. Eggs were elliptical with one side flattened, containing a knob in the anterior end, double thin walled and a developing embryo, and measuring 0.080 mm (0.075–0.088 mm) in length, and 0.034 mm (0.034–0.035 mm) in width (Figs. 4A, 4D, 5D, and 6F).

Oviducts empty into the straight transparent uterus (Fig. 6D) with its characteristically pointed end. The uterus continues as a straight tube with the vagina (Fig. 6D), which opens at the genital opening (Fig. 6D). Parallel to the uterus is a long tubule that connects to the vagina and extends posteriorly, ending in a convoluted mass in the anus region. From its position and structure, this tubule appears to be a rudimentary uterus and oviduct. The tail exhibits fine striations over the entire length, even showing through the annular bands and measuring 0.828 mm (0.579–0.945 mm) in length (Figs. 4A, 5A, 5C, 6D, and 6E, and table 2).

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 fine 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 amplified and sequenced for Passalurus ambiguus belonging to the families of Taeniidae Ludwig, 1886. The PCR amplification ranged from 700 to 800 bp. The sequence data of Passalurus ambiguus (781 nucleotides) examined were deposited in GenBank with accession numbers MZ571165.

The obtained sequences were aligned with 12 reference sequences representing the Oxyuroidea available species (Table 3); three species of Thelandros (T. galloti, T. tinerfensis, and T. filiformis); two species of Pharyngodon (P. micipsae and P. echinatus) and Skrjabinema (S. ovis and S. longicaudatum); one species of Batracholandros (B. salamandrae), Passalurus (P. ambiguus), Oxyuris (O. equis), Heteroxynema (H. cucullatum), and Aspicularis (A. tetraptera) together with Ascaridia galli (Ascaridiidae) as an outgroup. Thus, all sequences (including outgroup) were aligned over 872 positions.

The phylogenetic analysis was done using ML and MP methods. The obtained phylogenetic trees are shown in Figs. 9 and 10.

Phylogenetic trees based on the partial 28S sequence data showed that the superfamily Oxyuroidea was divided into four monophyletic clades, representing three families. Family Pharyngodonidae included genera of Pharyngodon (P. micipsae and P. echinatus), Thelandros (T. galloti, T. tinerfensis, and T. filiformis) and Batracholandros (B. salamandrae). Family Oxyuridae included genera of Passalurus (P. ambiguus), Oxyuris (O. equis), and Skrjabinema (S. ovis and S. longicaudatum), and were divided into two monophyletic clades. Family Heteroxynematidae included Heteroxynema (H. cucullatum) and Aspicularis (A. tetraptera).

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. filiformis 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 findings

The macroscopic appearance of the appendix was enlarged and filled 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 inflammatory cells against the worm infestation, with inflammatory 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).

Discussion

Oxyurids are nematode parasites that are found all over the world and have serious public health consequences (Abdel-Gaber, 2016; Khalil et al., 2014). Passalurus species (Oxyuridae) have been found in domestic and wild rabbits, but only a few research have concentrated on these nematodes (Millazzo et al., 2010; Robles and Navone, 2007; Sotillo et al., 2012). Passalurus ambiguus Rudolphi, 1819, P. nonanulatus Skinker, 1931, P. abditus Caballero, 1937, P. parvus Johnston and Mawson, 1938, and P. assimitis Wu, 1933 are the five species in this genus. Passalurus ambiguus is an ambiguous species of Passalurus, and it was originally described in the Palearctic region among Oryctolagus cuniculus and Lepus europeus Hall, 1916. Passalurus ambiguus is the most frequent intestinal worm found in Egyptian rabbits. The present species share morphological features with Passalurus, including the triangular mouth opening provided by four papillae and three teeth; the shape and size of the spicule; the lack of the gubernaculum and the location of the male cloacal papillae (Dos Santos et al., 2017; Hugot et al., 1983; Petter and Quentin, 2009; Sultan et al., 2015; Vicente et al., 1997; Yamaguti, 1961). Based on the mentioned factors, the current species has been identified as P. ambiguus. The current species were morphologically and morphometrically compared to that obtained by Abdel-Gaber et al. (2019) and Rodriguez et al. (1974), and it was found to be strikingly similar to P. ambiguus. The investigation by Abdel-Gaber et al. (2019) and Rodriguez et al. (1974) revealed similar results, with only minor changes in body part measurements.

Bin and Chunsheng (1987) mentioned the presence of lips in P. ambiguus in a brief comment, despite the fact that practically all known keys (Hugot et al., 1983; Vicente et al., 1997; Petter and Quentin, 2009) 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.

In males, SEM gave a clear view of the cloacal region topography. There are three papillae pairs (the first two pairs were pericloacal and larger than the last pair, which was small, sessile, and barely postcloacally located.). This finding 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) confirmed 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. (2015) and Abdel-Gaber et al. (2019). Based on the criteria provided, the results show that the female samples collected here are those of P. ambiguus.

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 classified 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 confirmed by the current phylogenetic analyses. Thefindings 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 first 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 findings of Li et al. (2019) and Cao et al (2020).

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 significant support value (BI = 100). This may be explained due to insufficient 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 finding, 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 inflammation 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 findings of acute or chronic inflammation in pinworm-infested appendix specimens. However, most studies show that appendiceal pinworms cause fewer inflammatory 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 first focusing on the presence of P. ambiguus in the appendix of domestic rabbits with histopathological examination. In conclusion, the current findings indicate that the pinworm species infecting Egyptian domestic rabbits is P. ambiguus Rudolphi, 1819.

Declarations

Acknowledgments

The authors would like to thank the Electron Microscopy Unit at South Valley University for their direct help and technical assistance with scanning electron microscopic investigations and EKB editing service for English editing of manuscript.

Author contribution

Nermean M. Hussein: investigation, methodology, writing—review and editing. Soheir A. H. Rabie: supervision, writing—review and editing. Wafaa A. Abuelwafa: collecting samples, methodology, writing—original draft. Mouchira M. Mohi ElDin:, writing—review and editing the histopathological part.

Funding

Not applicable.

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.

Conflict of interest

The authors declare no competing interests.

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Tables

Table (1): Estimation of evolutionary divergence between the present sequences of Passalurus ambiguus and the previously recorded sequences of Oxyuroidea.

Table (2): Comparative measurements (mm) of Passalurus ambiguus Rudolphi (1819) with those previously described.

Table (3): Partial 28S rDNA sequences of oxyuroidean species used in this study with their host species, locality and GenBank accession numbers. (*sequences collected from present study).