Morphological Re-Description and 18 S rDNA Sequence Confirmation of the Pinworm Aspiculuris tetraptera (Nematoda, Heteroxynematidae) Infecting the Laboratory Mice Mus musculus

A total of 28 out of 50 (56.0%) specimens of laboratory mice M. musculus were found to be naturally infected with an oxyurid nematoda. The recovered parasite species were found in the cecum and upper colon of the infected host mice.

Description

In general, the body of the recovered worms was small, cylindrical in shape, and covered by a transversely striated cuticle. Head was bulb-like; mouth opening was surrounded by three less developed lips, one pair of lateral epaulettes, one pair of amphidial pores, and two pairs of large sub-median cephalic papillae. Mouth opening leads to the buccal cavity, followed by pharynx, esophagus, and long intestine opening exteriorly by an anal opening in females and cloacal opening in males. Anterior part of esophagus was club-shaped followed by well-developed bulb. Body in both sexes has distinct cervical alae, beginning immediately posterior to the anterior end of the cephalic vesicle. Cervical alae abruptly interrupted at the level of esophago–intestinal junction, forming an acute angle. Anterior end of the body has prominent, elaborate inflated region, forming cephalic vesicle (Fig. 1A–G, Tables 2,3).

Table 2

Main morphological features and measurements of male Aspiculuris tetraptera compared with previous studies.

Related species	Aspiculuris ackerti	Aspiculuris artigasi	Aspiculuris versterae	Aspiculuris tetraptera	A. ackerti	A. tetraptera	A. tetraptera	A. tetraptera	Aspiculuris huascaensis	Aspiculuris tianjinensis	A. tetraptera	A. tetraptera	A. tetraptera
Parameters	Kruidenier and Mehra (1959)	Araujo (1965)	Hugot (1980)	Hugot (1980)	Miller and Schmidt (1982)	Kohn and Macedo (1984)	Pinto et al. (1994)	Landaeta-Aqueveque et al. (2007)	Falcón-Ordaz et al. (2010)	Liu et al. (2012)	Khalil et al. (2014)	Abdel-Gaber and Fol (2015)	(Present study)
Host species	Neotoma albigtda	M. musculus	Mastomys natalensis	Mastomys natalensis	Neotoma cinerea	Haemulon sciurus	M. musculus	M. musculus	M. musculus	Clethrionomys rufocanus	M. musculus	M. musculus	M. musculus
Host locality	Coconimo, Arizona	Sao Paulo, Brazil	Onderstepoort, South Africa	Maracay, Venezuela	Colorado, Idaho, USA	Rio de Janeiro, Brazil	Río de Janeiro, Brazil	Santiago, Chile	Hidalgo, Mexico	Tianjin, China	Cairo, Egypt	Cairo, Egypt	Cairo, Egypt
Site of infection	Intestine	Intestine	Caecum	Caecum	Caecum	Intestine	Intestine	Caecum and upper colon	Intestine	Intestine	Caecum	Caecum	Caecum and upper colon
Body length	4.41–4.87 (4.64)	2.945–3.472	1.59	2.7	3.01–6.35 (5.03)	2.90–3.60 (3.22)	2.4–3.1	2.65–3.95 (3.393 ± 0.427)	2.16–2.69 (2.48 ± 0.172)	3.69– 5.12 (4.69)	2.55–2.57 (2.56 ± 0.014)	2.73–3.87 (3.21 ± 0.1)	2.23–3.29 (2.79 ± 0.1)
Body width	0.162–0.174 (0.168)	0.156–0.180	0.10	0.11	0.137–0.293 (0.236)	0.15–0.20 (0.17)	0.17–0.21	0.17–0.25 (0.202 ± 0.2)	0.078–0.114 (0.096 ± 0.01)	0.204–0.388 (0.307)	0.088 (0.088 ± 0)	0.18–0.23 (0.20 ± 0.1)	0.16–0.20 (0.18 ± 0.1)
Esophageal length	0.264–0.271 (0.267)	0.213–0.243	0.310	0.38	0.260–0.480 (0.343)	0.18–0.27 (0.23)	0.288–0.360	0.320–0.390 (0.375 ± 0.02)	0.291–0.294 (0.272 ± 0.013)	0.343–0.397 (0.384)	0.439–0.450 (0.44 ± 0.007)	0.38–0.45 (0.41 ± 0.1)	0.32–0.40 (0.39 ± 0.1)
Esophageal width	0.039–0.040 (0.039)	0.036–0.054	–	–	0.033–0.060 (0.051)	–	–	–	–	–	–	0.06–0.11 (0.08 ± 0.01)	0.05–0.09 (0.07 ± 0.01)
Esophageal bulb length	0.145–0.155 (0.150)	0.105–0.135	0.10	0.14	0.113–0.183 (0.157)	0.10-0.12 (0.11)	–	0.110–0.140 (0.133 ± 0.01)	0.090–0.110 (0.097 ± 0.006)	0.074–0.142 (0.122)	0.244 (0.24 ± 0)	0.14–0.19 (0.16 ± 0.1)	0.13–0.17 (0.15 ± 0.1)
Esophageal bulb width	0.071–0.079 (0.075)	0.075–0.096	0.08	0.09	0.065–0.120 (0.090)	0.06–0.08 (0.07)	–	–	0.040–0.063 (0.051 ± 0.006)	0.059–0.123 (0.098)	0.077–0.088 (0.08 ± 0.007)	0.07–0.09 (0.08 ± 0.01)	0.04–0.07 (0.05 ± 0.01)
Distance from anterior extremity Nerve ring	0.158	0.108–0.144	0.09	0.10	0.150–0.213 (0.186)	0.11–0.13 (0.12)	0.115–0.140	0.115–0.145 (0.129 ± 0.013)	0.072–0.093 (0.080 ± 0.008)	0.116–0.133 (0.123)	0.088 (0.088 ± 0)	–	0.065–0.082 (0.078 ± 0.001)
Excretory pore	–	0.600–0.876	0.325	0.78	0.69–1.21 (0.96)	0.69–0.78 (0.73)	0.619	0.750 –0.910 (0.854 ± 0.056)	0.495–0.579 (0.545 ± 0.035)	0.93–1.03 (1.01)	0.144–0.147 (0.145 ± 0.002)	–	0.392–0.547 (0.491 ± 0.03)
No. of Caudal papillae	11	5	5	5	10	12	12	12	12	12	12	12	12
Tail length	0.238–0.280 (0.259)	0.147–0.183	0.12	0.17	0.049–0.117 (0.092)	0.10-0.12 (0.11)	0.144–0.172	0.162–0.187 (0.178 ± 0.009)	0.075–0.114 (0.102 ± 0.013)	0.181–0.245 (0.220)	0.12–0.144 (0.132 ± 0.01)	0.13–0.15 (0.14 ± 0.1)	0.11–0.14 (0.12 ± 0.1)

Table 3

Main morphological features and measurements of female Aspiculuris tetraptera compared with previous studies.

Related species	Aspiculuris ackerti	Aspiculuris artigasi	Aspiculuris versterae	Aspiculuris tetraptera	Aspiculuris ackerti	A. tetraptera	A. tetraptera	A. tetraptera	Aspiculuris huascaensis	Aspiculuris tianjinensis	A. tetraptera	A. tetraptera	A. tetraptera
Parameters	Kruidenier and Mehra (1959)	Araujo (1965)	Hugot (1980)	Hugot (1980)	Miller and Schmidt (1982)	Kohn and Macedo (1984)	Pinto et al. (1994)	Landaeta-Aqueveque et al. (2007)	Falcón-Ordaz et al. (2010)	Liu et al. (2012)	Khalil et al. (2014)	Abdel-Gaber and Fol (2015)	(Present study)
Host species	Neotoma albigtda	Mus musculus	Mastomys natalensis	M. natalensis	Neotoma cinerea	Haemulon sciurus	M. musculus	M. musculus	M. musculus	Clethrionomys rufocanus	M. musculus	M. musculus	M. musculus
Host locality	Coconimo, Arizona	Sao Paulo, Brazil	Onderstepoort, South Africa	Maracay, Venezuela	Colorado, Idaho, USA	Rio de Janeiro, Brazil	Río de Janeiro, Brazil	Santiago, Chile	Hidalgo, Mexico	Tianjin, China	Cairo, Egypt	Cairo, Egypt	Cairo, Egypt
Site of infection	Intestine	Intestine	Caecum	Caecum	Caecum	Intestine	Intestine	Caecum and upper colon	Intestine	Intestine	Caecum	Caecum	Caecum and upper colon
Body length	5.5–6.8 (6.2)	3.534–5.394	1.95	1.2	5.46–8.97 (7.41)	3.60–4.61 (4.18)	3.1–3.6	3.53–4.51 (3.53–4.51)	3.024–3.528 (3.219 ± 0.150)	5.38–7.00 (6.46)	3.55–4.12 (3.83 ± 0.403)	3.2–3.6 (3.5 ± 0.1)	2.9–3.4 (3.1 ± 0.1)
Body width	0.197–0.232 (0.210)	0.153–0.234	0.15	0.08	0.225–0.391 (0.330)	0.20–0.30 (0.27)	0.175–0.245	0.230–0.270 (0.260 ± 0.01)	0.099–0.153 (0.120 ± 0.016)	0.282–0.447 (0.352)	0.199–0.288 (0.244 ± 0.062)	0.21–0.26 (0.24 ± 0.01)	0.19–0.23 (0.20 ± 0.01)
Esophageal length	0.290–0.348 (0.317)	0.252–0.336	0.49	0.43	0.305–0.465 (0.387)	0.25–0.29 (0.27)	0.331–0.433	0.400–0.450 (0.425 ± 0.01)	0.312–0.351 (0.322 ± 0.012)	0.417–0.480 (0.461)	0.328–0.382 (0.35 ± 0.038)	0.33–0.39 (0.27 ± 0.01)	0.30–0.34 (0.32 ± 0.01)
Esophageal width	0.042–0.052 (0.044)	0.048–0.057	–	–	0.045–0.080 (0.061)	–	–	–	–	–	–	0.17–0.19 (0.18 ± 0.01)	0.14–0.16 (0.15 ± 0.01)
Esophageal bulb length	0.145–0.171 (0.158)	0.129–0.159	0.14	0.16	0.150–0.225 (0.177)	0.12–0.15 (0.13)	–	0.140–0.160 (0.152 ± 0.006)	0.090–0.111 (0.103 ± 0.005)	0.123–0.167 (0.152)	0.108 (0.108 ± 0)	0.12–0.15 (0.140 ± 0.01)	0.10–0.13 (0.11 ± 0.01)
Esophageal bulb width	0.068–0–0.087 (0.080)	0.087–0.111	0.095	0.13	0.080–0.150 (0.107)	0.08–0.12 (0.10)	–	–	0.054–0.087 (0.063 ± 0.008)	0.088–0.137 (0.118)	0.081 (0.081 ± 0)	0.07–0.11 (0.09 ± 0.01)	0.05–0.09 (0.07 ± 0.01)
Distance from anterior extremity Nerve ring	0.155–0.185 (0.168)	0.120–0.168	0.14	0.13	0.162–0.238 (0.202)	0.15	0.158–0.216	0.12–0.162 (0.140 ± 0.013)	0.081–0.0105 (0.093 ± 0.007)	0.121–0.157 (0.134)	0.144–0.147 (0.145 ± 0.002)	–	0.078–0.090 (0.085 ± 0.002)
Excretory pore	–	0.756–1.056	0.53	0.9	0.82–1.67 (1.10)	0.81–0.89 (0.86)	0.734–0.936	0.860–0.950 (0.888 ± 0.032)	0.555–0.675 (0.590 ± 0.038)	1.09–1.23 (1.17)	–	–	0.564–0.780 (0.680 ± 0.02)
Vulval opening	2.1–2.5 (2.3)	1.457–2.015	0.85	1.65	1.52–2.89 (2.49)	1.54–2.36 (1.76)	1.1–1.4	1.61  ± 0.038 (1.55–1.67)	1.204–1.428 (1.269 ± 0.066)	2.07–2.43 (2.27)	1.56–1.86 (1.71 ± 0.21)	–	1.112–1.630 (1.406 ± 0.03)
Tail length	0.232–0.690 (0.445)	0.444–0.660	0.435	0.55	0.088–0.110 (0.101)	0.36–0.40 (0.39)	0.360–0.486	0.347–0.537 (0.477 ± 0.06)	0.330–0.372 (0.358 ± 0.016)	0.92–1.14 (1.05)	0.382–0.405(0.393 ± 0.016)	0.34–0.48 (0.42 ± 0.01)	0.30–0.42 (0.39 ± 0.01)
Eggs length	0.097–0.106 (0.101)	0.084–0.090	0.09	0.095	0.093–0.110 (0.102)	0.075–0.090 (0.084)	0.72–0.90	0.084–0.092 (0.087 ± 0.005)	0.054–0.069 (0.061 ± 0.003)	0.092–0.096 (0.094)	0.115–0.129 (0.122 ± 0.008)	0.05–0.07 (0.06 ± 0.01)	0.04–0.06 (0.05 ± 0.01)
Eggs width	0.0390.047 (0.044)	0.036–0.039	0.04	0.045	0.038–0.055 (0.045)	0.038–0.051 (0.045)	0.036 –0.053	0.043–0.052 (0.046 ± 0.005)	0.018–0.030 (0.022 ± 0.003)	0.051–0.058 (0.054)	0.054–0.072 (0.06 ± 0.008)	0.03–0.05 (0.04 ± 0.01)	0.02–0.04 (0.03 ± 0.01)

Male worm (based on 10 mature specimens)

Body length was 2.23–3.29 (2.79 ± 0.1) mm with maximum width 0.16–0.20 (0.18 ± 0.1) mm. Cephalic vesicle was 0.06–0.09 (0.07 ± 0.001) mm long by 0.05–0.08 (0.06 ± 0.001) mm wide. Esophagus measured 0.32–0.40 (0.39 ± 0.1) mm long by 0.05–0.09 (0.07 ± 0.01) mm wide; while, the whole esophagus with bulb reached 0.13–0.17 (0.15 ± 0.1) mm long by 0.04–0.07 (0.05 ± 0.01) mm wide. Cervical alae began at 0.015–0.018 (0.017 ± 0.001) mm from the anterior end and measured about 0.21–0.29 (0.25 ± 0.001) mm long with recurved terminal ends by 0.029–0.038 (0.031 ± 0.001) mm wide. Nerve ring and excretory pore are located at 0.065–0.082 (0.078 ± 0.001) mm and 0.392–0.547 (0.491 ± 0.03) mm from the anterior end, respectively. Narrow lateral alae of the body end located at the beginning of the caudal alae are extended from the level of cloaca and surrounded the entire end of the body, bending ventrally at its tip as a vesicular swelling of the cuticle. Cloaca opening is located at 0.09–0.11 (0.10 ± 0.01) from the posterior extremity of the body. Testes are flexed over the anterior third of the intestine. Gubernaculum and spicules were absent. Posterior end with 12 caudal papillae included one pair precloacal, two pairs adcloacal, one pair postcloacal, two median papillae postcloacal, one behind the other, and a further posterior pair midway between cloaca and end of the tail. Tail, with blunt end, measured 0.11–0.14 (0.12 ± 0.1) mm long.

Fig. 1

Female worm is larger than that of the male (based on 10 mature specimens)

Body length was 2.9–3.4 (3.1 ± 0.1) mm long with maximum width was 0.19–0.23 (0.20 ± 0.01) mm. Cephalic vesicle reached about 0.078–0.083 (0.082 ± 0.001) mm long by 0.106–0.130 (0.123 ± 0.01) mm wide. Esophagus measured 0.30–0.34 (0.32 ± 0.01) mm long and 0.14–0.16 (0.15 ± 0.01) mm wide; while, esophagus with bulb reached about 0.10–0.13 (0.11 ± 0.01) mm long and 0.05–0.09 (0.07 ± 0.01) mm wide. Nerve ring and excretory pore located at 0.078–0.090 (0.085 ± 0.002) mm and 0.564–0.780 (0.680 ± 0.02) mm from the anterior end, respectively. Cervical alae with recurved terminal end was 0.27–0.29 (0.26 ± 0.01) mm long. Distance from the anterior end to the beginning of cervical alae was 0.021–0.026 (0.024 ± 0.001) mm. Vulva was preequatorial, surrounded by protruded lips, and situated at 1.112–1.630 (1.406 ± 0.03) mm from the anterior extremity of the body. Ovejector apparatus measured about 0.29–0.38 (0.32 ± 0.01) mm long. Muscular vagina proceeded forward for a short distance then turned backward joining uterus filled with eggs. Two ovaries flexed over the proximal part of the intestine. Anal pore located at 0.32–0.39 (0.37 ± 0.01) mm from the posterior end of the body. Tail with blunt tip measured 0.30–0.42 (0.39 ± 0.01) mm long. Eggs were unoperculated, smooth, filled by morula and measured 0.04–0.06 (0.05 ± 0.01) mm long and 0.02–0.04 (0.03 ± 0.01) mm wide.

Molecular analysis

A sequence of 840 bp was deposited in GenBank under accession no. MG019400 with a GC content of 42.26%, for SSU rDNA gene sequences of the present oxyurid species. Pairwise comparison of the isolated gDNA sequence of the present parasite species with a range of other Spirurina species and genotypes revealed a unique sequence. The calculated identity between this novel sequence and those retrieved from GenBank demonstrated a high degree of similarity, up to 88.0% (Table 1). Comparison of the nucleotide sequences and divergence showed that SSU rDNA of the present oxyruid species had the highest blast scores with a small number of nucleotide differences with other A. tetraptera species under the following accession numbers: EU263107, EF464551, KJ143616, KJ143617, KJ143618, KJ143615, KT175729, KT175728, KT175725, and KT175725; A. dinniki (acc. no. KT175736); A. tianjinensis (acc. no. KT175733); E. vermicularis (acc. no. HQ646164); and S. kamosika (acc. no. AB699691).

Phylogenetic analysis led to the construction of a neighbor-joining tree, constructed with partial sequences, which showed that Spirurina species consistently formed two major clades (Fig. 2). The first one represented the most related families in order Oxyurida, including families Heteroxynematidae and Oxyuridae with sequence similarity ranging between 99.0 and 91.0%. The second one was represented by four families Ascarididiae (P. equorum JN617987), Anisakidae (P. rajae JN392470), Cucullanida (C. extraneus KT192060), and Cosmocercidae (C. japonica LC052782), belonging to the order Ascaridia with sequence similarity ranging between 90.0 and 89.0%. This sequence in conjunction with existing data, suggested the placement of this oxyurid species within family Heteroxynematidae. The present species was deeply embedded in the genus Aspiculuris, and is closely related to other Aspiculuris species, especially to other previously described A. tetraptera as a putative sister taxon.

Fig. 2

Laboratory animal models, especially rodents of the family Muridae, constitute important links in the food chains within the ecosystems they inhabit (Rosas, 1997, Gonçalves et al., 1998). These animals are often in contact with humans and domestic animals and can transmit various parasitic species (Bazzano et al., 2002). In conventional animal facilities, rodent colonies are frequently infected with helminth parasites, or become infected during the experimental period (Sato et al., 1995, Rehbinder et al., 1996). Oxyurids are cosmopolitan nematoda parasites of public health importance (Khalil et al., 2014). The order Oxyurida includes three families namely Oxyuridae (Cobbold, 1864); Pharyngodonidae (Travassos, 1919); and Heteroxynematidae (Skrjabin and Schikhobalova, 1948). Nematodes from the genera Syphacia and Aspiculuris are common parasitic pinworms of rodents all over the world (Adamson, 1994; Robles and Navone, 2007; Millazzo et al., 2010; Sotillo et al., 2012; Verma et al., 2013; Weyand et al., 2016; Zarei et al., 2016; Stewart et al., 2017).

Based on morphological characters, the oxyurid species described here showed the characteristic features of the genus Aspiculuris, including the presence of four distinct cephalic papillae lying on the cephalic plate, and three small rudimental lips that carry two sessile poorly developed labial papillae. According to the present results, A. tetraptera naturally infect the laboratory mice M. musculus, which is consistent with data reported by Hugot (1988), Mohd Zain et al. (2012) Pakdel et al. (2013), and Panti-May et al. (2015) who stated that species of the genus Aspiculuris belonging to the family Heteroxynematidae are characterized as parasites of Muroidea. Based on the stated characters herein, the present species was identified as A. tetraptera and recorded in 56.0% of the examined specimens representing a higher prevalence. This is in agreement with data obtained by Bluszcz et al. (1987), Bazzano et al. (2002), Izdebska and Rolbiecki (2006), Klimpel et al. (2007), Kataranovski et al. (2008), and Baird et al. (2012), who reported that mice are naturally infected with different oxyurid species, with 9.0 to 75.0% infection range.

The present parasite species was compared morphologically and morphometrically with other Aspiculuris species as shown in Tables 2 and 3, and exhibited strong similarities to those reported in other studies by Yamaguti (1935), Hugot (1980), Pinto et al. (1994), Landaeta-Aqueveque et al. (2007), Khalil et al. (2014), and Abdel-Gaber and Fol (2015). Only a few differences in measurements of the different body parts were observed. However, this species differs from other Aspiculuris species in the structure of the cephalic region, length of esophagus, position of nerve ring, excretory pore and vulva opening, number and arrangement of cloacal papillae in males, and size of eggs in females. The presently described parasite species resembled A. tetraptera and Aspiculuris huascaensis by having cervical alae ending at the mid-length of the esophageal bulb with the same number of caudal papillae. However, it can be distinguished from A. huascaensis by having a single sessile pre-cloacal papilla located between two cuticular folds and slightly anteriorly to the cloaca, whereas A. tetraptera lack both the sessile pre-cloacal papilla and two cuticular folds. In addition, males of A. tetraptera have a double pedunculate papilla immediately posterior to the cloaca, and the anterior most papilla located between the caudal folds of the tail is double, while, those of A. huascaensis lack a doubled medial papilla associated with the cloaca and a simple anterior most papillae was recorded. These data were consistent with a previous report by Falcón-Ordaz et al. (2010). In agreement with Ashour (1980), Pinto et al. (1994), and Abdel-Gaber and Fol (2015), 12 papillae were present in A. tetraptera; conversely, Schulz (1924) reported 10 and Yamaguti (1935) and Falcón-Ordaz et al. (2010) reported 14 papillae. In addition, the division of the dorsal and both subventral lips of A. tetraptera males are unique. Chitwood and Chitwood (1950) noted a similar division of the dorsal lip only of A. ackerti with sexual dimorphism of this character in male worms and no tendency for separation in the female specimens.

Aspiculuris is one of five subgenera listed by Akhtar (1955) in which the cephalic bulb and lateral alae are present, the cervical alae end in a sickle shaped margins, but the cervical and lateral alae are not continuous, as stated by Petter and Quentin (2009). The same results were obtained in the present study on the recovered worms, which have well-developed cervical alae extending into cephalic vesicle, and poorly marked cuticular striations. Furthermore, the present described parasite species were similar to other species of Aspiculuris, such as Aspiculuris dinnicki (Schulz, 1924); Aspiculuris schulzi (Popov and Nasarova, 1930); Aspiculuris azerbaidjanica (Tarzhimanova, 1969); Aspiculuris arianica (Erhadová-Kotrlá and Daniel, 1970); and Aspiculuris witenbergi (Quentin, 1975); with cervical alae that are abruptly interrupted with the pointed posterior ends and forming an acute angle toward the anterior. However, it differs from Aspiculuris kazakstanica (Nasarova and Sweschikova, 1930); Aspiculuris americana (Erickson, 1938); Aspiculuris lahorica (Akhtar, 1955); Aspiculuris pakistanica (Akhtar, 1955); Aspiculuris africana (Quentin, 1966); Aspiculuris tschertkowi (Tarzhimanova, 1969); Aspiculuris rysavyi (Kotrla and Daniel, 1970); and Aspiculuris versterae (Hugot, 1980), since the posterior end of the cervical alae of those species does not form an acute angle, and the caudal alae of males is not close to the tip. In addition, with the exception of A. tschertkowi, which has 16 caudal papillae, the remaining species have a smaller number of papillae than A. huascaensis, varying from 4 to 11 versus 12 papillae.

Due to close morphological similarities, molecular phylogenetic approaches have been used extensively in association with traditional morphological techniques as reliable methods for confirmation of accurate identification, and differentiation between pinworms infecting laboratory rodents (Jacobs et al., 1997; Zhu et al., 1998; Vermund and Wilson, 2000; Morales-Hojas et al., 2001; Nakano et al., 2006; Li et al., 2007; Zhu et al., 2007; Chang et al., 2009). In the present study, a nuclear rDNA region of the recovered parasite species was amplified using the species-specific primers Nem 18SF/Nem 18SR, designed by Floyd et al. (2005). It is apparent that, the phylogenetic tree based on nuclear SSU rDNA sequences estimated in this study supported strongly the higher taxonomic groups of both orders: Oxyurida (representing the two main families Oxyuridae and Heteroxynematidae) and Ascaridia (representing four families, namely Cosmocercidae, Cucullanidae, Anisakidae, and Ascarididiae). These results are in agreement with data obtained by Blaxter et al. (1998) who reported that clade III of the full dataset of the nematoda phylogeny was represented by all members of the suborder spirurina and clustered into four classical orders of Ascaridia, Oxyurida, Rhigonematida, and Spirurida. Anderson (2000) proposed that Ascaridia and Spirurida were sister groups, which in turn, were more closely related to Strongylida than a group consisting of Oxyurida plus Rhigonematida. Subsequent analyses of SSU rDNA sequences strongly supported the monophyly of clade III taxa with bootstrap values for the clade exceeding 95.0% (De Ley and Blaxter, 2002; Bert et al., 2006; Holterman et al., 2006; Wijová et al., 2006; Qiu et al., 2016; Ribas et al., 2017).

Khalil et al. (2014) reported that the order Oxyurida incorporates three main families, Oxyuridae (Cobbold, 1864); Pharyngodonidae (Travassos, 1919); and Heteroxynematidae (Skrjabin and Schikhobalova, 1948); which was consistent with the results of the current study. In addition, Petter and Quentin (2009) included Syphacia obvelata and Syphacia muris of the genus Syphacia with 22 genera in the family Oxyuridae, and included the genus Aspiculuris together with seven further genera in the subfamily Heteroxynematinae belonging to the family Heteroxynematidae. This was consistent with the present findings indicating that Oxyuridae species, represented by the genus Aspiculuris, is monophyletic in origin, supporting the taxonomic position of the present Aspiculuris species, which is deeply embedded in the genus Aspiculuris with a close relationship with other described species of A. tetraptera as a more related sister taxon.

Recent field studies have provided useful tools for the rapid identification and phylogenetic analysis of pinworms infecting laboratory rodents. The 18S rDNA gene of A. tetraptera yielded a unique sequence that confirms the taxonomic position within the family Heteroxynematidae.

All procedures contributing to this work comply with the ethical standards of the relevant national guides on the care and use of laboratory animals and have been approved and authorized by Institutional Animal Care and Use Committee (IACUC) in Faculty of Science, Cairo University, Egypt (No. CU/I/S/19/16).