JOURNAL of NEMATOLOGY Morphological Re
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JOURNAL OF NEMATOLOGY Article | DOI: 10.21307/jofnem-2018-026 Issue 0 | Vol. 0 Morphological Re-Description and 18 S rDNA Sequence Confirmation of the Pinworm Aspiculuris tetraptera (Nematoda, Heteroxynematidae) Infecting the Laboratory Mice Mus musculus Rewaida Abdel-Gaber,1,2* Fathy Abdel-Ghaffar,2 Saleh Al Quraishy,1 Kareem Morsy,2,3 Rehab Saleh4 Abstract and Heinz Mehlhorn5 Aspiculuris tetraptera is a heteroxynematid nematoda infecting most 1Zoology Department, College of of the laboratory animals, occasionally mice which represent the Science, King Saud University, mostly used animal for biological, medical, and pharmacological Riyadh, Saudi Arabia. studies. The present study aimed to investigate the prevalence of nematode parasites infection in the laboratory mice Mus musculus in 2 Faculty of Science, Zoology Egypt. Morphologically, this oxyurid possessed four distinct cephalic Department, Cairo University, papillae on a cephalic plate, with three small rudimental lips carrying Cairo, Egypt. two sessile poorly developed labial papillae and one pair of amphidial 3Biology Department, College of pores. Esophagus divided into cylindrical corpus and globular bulb. Science, King Khalid University, Distinct cervical alae interrupted at the level of esophago–intestinal Abha, Saudi Arabia. junction forming an acute angle. At the caudal end, twelve caudal papillae in male worms while an ovijector apparatus opening and a 4 Faculty of Arts and Sciences, vulva surrounded by protruded lips in females were observed. The Biology Department, Kasr-Khiar, general morphological criteria include this nematode with other Al Mergheb University, Libya. Aspiculuris species which were compared in the present study. 5Parasitology Institute, Düsseldorf Molecular characterization based on 18SSU rDNA sequencing University, Düsseldorf, Germany. performed to confirm the taxonomic position of this species and to documents the morphological data. Sequence alignment detects *E-mail: [email protected] a percent of identity up to 88.0% with other Heteroxynematidae & [email protected] species. Phylogenetic analysis showed that the present recorded is a This article was edited by Zafar putative sister taxon to A. tetraptera recorded in a previous study. The Handoo. SSU rDNA sequence has been deposited in the GenBank under the accession no. MG019400. Received for publication February 10, 2018. Key words Laboratory mice, Pinworms, Aspiculuris species, Morphological description, Molecular study Pinworms are routinely found in animals from modern et al., 2004; Robles and Navone, 2010; Khalil et al., animal facilities, even in facilities free of viral and bac- 2014). Mice may be concurrently infected with both terial diseases that affect mice (Jacoby and Lindsey, species of pinworms (Jacobson and Reed, 1974; Taf- 1998; Zenner and Regnault, 2000; Behnke et al., fs, 1976; Nicklas et al., 1984; Gonçalves et al., 1998; 2015). Oxyurids are also a common parasite of Zenner, 1998; Agersborg et al., 2001). The common Muroidea (Rodentia) (Singleton et al., 1993; Pisanu incidence of infection by both pinworm species can be et al., 2001). Syphacia obvelata and Aspiculuris explained by their preference for slightly different sites tetraptera are oxyurid nematodes which are cosmo- of the gastrointestinal tract. As such, these species politan monoxenous parasites that are transmitted do not compete directly for resources; they are able through the ingestion of embryonated eggs (Stojcevic to maintain simultaneous infections (Pinto et al., 2001; 1 © The Society of Nematologists 2018. Morphological Re-Description and 18 S rDNA Sequence Confirmation of the Pinworm Aspiculuris tetraptera Bazzano et al., 2002). In concurrent infections, there Materials and Methods may be higher numbers of A. tetraptera worms be- cause their longer lifespan permits the accumulation of Animal collection and parasitological parasites in their hosts (Scott and Gibbs, 1986). The examination prevalence of pinworms in an infected rodent popula- tion depends on many factors, including gender, age, Fifty specimens of adult laboratory mice (Muridae: strain, immune status, and the concentration of para- M. musculus) reared at the Animal House at Zoology site ova in the environment. Department, Faculty of Science, Cairo University, Cairo, The genus Aspiculuris was established by Egypt; were randomly collected between December Nitzsch (1821) and later re-described by Schulz 2016 and September 2017. The collected mice were (1924) from Mus musculus. Many species of As- transported alive to the Laboratory of Parasitology piculuris have been reported worldwide (Hugot, Research for parasitological examination. Mice were 1980; Inglis et al., 1990; Falcón-Ordaz et al., 2010). anesthetized and killed according to the ethical rules for Species of Aspiculuris were separated by Quentin handling experimental animals. Mice were examined for (1975) into two groups based on the shape of their any external signs of infection. After dissection, internal cervical alae. Nematodes that display interrupted organs were removed from the rodent and examined cervical alae with pointed posterior ends belong to for any parasitic infections. Isolated worms were fixed in the first group. Those in the second group possess 70% ethanol and subsequently clarified with lactophe- a rounded posterior end to the cervical alae. As- nol for morphological identification, in accordance with picularis tetraptera are common oxyurids belonged standard reference keys by Pinto et al. (2001). Preva- to the first group. This species has been described lence of parasitic infection (number of infected mice/ in the cecum and colon of M. musculus in different total number of mice hosts examined ×100) of M. mus- regions, such as Tunisia, Iran, Venezuela, Europe, culus was calculated according to Bush et al. (1997). Siberia, China, Japan, Unite States, and Egypt Illustrations of adult specimens were prepared with the (Hugot, 1980; Neifer et al., 1991; Durden et al., aid of a microscope Leica DM 2500, LAS software (3.8) 2000; Perec-Matysiak et al., 2006; Mahmoud et al., and Corel Draw X4® software. Measurements were 2009; Abdel-Gaber, 2016), and to a lesser extent, based on 20 adult worm species; data were taken in it has been recorded in other hosts, such as Cri- millimeters and are presented as a range followed by cetus, Rattus, Apodemus, Microtus, Arctomys, Jac- the arithmetic mean ± SD in parentheses. ulus, Clethrionomys, and Peromyscus in the same regions (Mathies, 1959; Sasa et al., 1962). Quen- tin (1975) indicated this species in Central Africa in Molecular analysis Mastomys, Praomys, and Thamnomys. Species of A. tetraptera are characterized by their DNA extraction, polymerase chain medium or small size, the presence of three lips, reaction amplification, and sequencing absence of buccal capsule, and presence of esoph- agus with a well-developed single bulb located at its gDNA was extracted from ethanol-preserved samples posterior end (Bazzano et al., 2002; Perec-Matysiak using DNeasy tissue kit© (Qiagen, Hilden, Germany) et al., 2006; Malsawmtluangi and Tandon, 2009, Li et following the manufacturer’s instructions. The DNA al., 2016). Recently, morphological identification re- was stored in 50 μ l of TE buffer at −20°C until further quires the use of molecular characteristics for accu- use. DNA concentration and purity were determined rate identification and validation; these characteristics spectrophotometrically by measuring absorbance at are common in nematoda systematics (Mc-Manus wavelengths of 260 and 280 nm. PCR amplification and Bowles, 1996; Semenova et al., 1996; Gasser, was performed in a final volume of 25 μl, containing 2001; Jones et al., 2012; Chaudhary et al., 2016; 3 μ l of genomic DNA, 2.5 μ l of 10X Taq polymerase Curtis et al., 2017). buffer, 10 pmol of each primer, 100 μ M of each dNTP Therefore, the present study reported the natu- (Finnzymes Products), and 1.5 U of Taq DNA poly- ral prevalence, morphological, and morphometric merase (Finnzymes Products). The partial ribosomal characteristics, in addition to molecular analysis of 18S gene was amplified using the primer Nem 18SF ribosomal DNA gene sequences of the recovered (5′-CGC GAA TRG CTC ATT ACA ACA GC-3′) and oxyurid pinworm infecting the laboratory mouse Nem 18SR (5′-GGG CGG TAT CTG ATC GCC-3′) de- M. musculus to clarify the taxonomic status and signed by Floyd et al. (2005). Polymerase chain reac- phylogenetic position of this parasite species within tion (PCR) consisted of an initial denaturation step at Heteroxynematidae. 94°C for 3 min, followed by 35 cycles of 1 min at 94°C, 2 JOURNAL OF NEMATOLOGY 1 min at 50°C, followed by 1 min at 72°C, and finally, ated cuticle. Head was bulb-like; mouth opening was post-PCR extension was carried out for 7 min at 72°C. surrounded by three less developed lips, one pair of All PCR products were verified on 1% agarose gel in lateral epaulettes, one pair of amphidial pores, and two ×1 Tris–acetate–EDTA (TAE) stained with 1% ethidium pairs of large sub-median cephalic papillae. Mouth bromide visualized with UV transilluminator, and bands opening leads to the buccal cavity, followed by phar- with predicted size were purified using Pure LinkTM ynx, esophagus, and long intestine opening exteriorly Quick Gel Extraction Kit (Invitrogen) following the man- by an anal opening in females and cloacal opening in ufacturer’s instructions. Amplicons were sequenced males. Anterior part of esophagus was club-shaped (in both directions)