MATERIALS AND METHODS

Study area: In 2015, for the purpose of identification of rabbit pinworms, visits of local slaughter-shops in Tanta City, Al-Gharbia Province, in the mid Delta of Egypt were accrued out. GITs of rabbits were collected and transferred to the laboratory as quickly as possible for examination. Preliminary ordinary microscopical identification, DNA extraction were done in Parasitology Lab, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt. Whereas, electron microscopic study was carried out in Electron Microscope Unit, Faculty of Science, Alexandria University, Egypt⁸.

Samples and identification: Gastrointestinal tracts (GITs) of domesticated rabbits were collected from local slaughter-shops of Tanta city, Al-Gharbia Province located in mid-Delta of Egypt. Collected gastrointestinal tracts were incised carefully, examined for presence of pinworms. Recovered specimens were washed thoroughly in 0.9% saline to remove host tissue debris and some were temporarily mounted on glass slides with/without the aid of lactophenol, for morphological identification of the species. Preservation of the specimens was done in either 10% neutral formalin (for morphological examination) or 70% ethanol (for molecular examination). For morphological identification of the species; the collected worms were examined under the ordinary microscope to show the characteristic identification characters like the double bulb-shaped esophagus, posterior ends of males and females. Also, to confirm the species a scanning electron microscopical examination was done for some specimens to see the topography of the surface and its characteristic features⁸.

DNA extraction: It was done by using commercial QIAamp^® DNA MiniKit (QIAGEN^®); the extraction followed the manufacturer’s recommendations. The extracted total genomic DNA served as a template in subsequent polymerase chain reaction (PCR).

PCR: Amplification of P. ambiguus partial cox1 and cyt b genes were done according to Sheng et al.⁹. Briefly; reactions were performed using the following primers pcox1 TTATGCCTATTATGATG (Forward), AACAACCAACTAAAAAC (Reverse), pcyt b TATTATGGGACTGATTT (Forward) ATAACAACACCTTTACA (Reverse).

The PCR cycling conditions were as follows: initial denaturation (enzyme activation) at 95°C for 2 min, followed by 35 cycles of 30 sec at 95°C for 1 min (denaturation), 1 min (annealing) at 47.9 "Cox11" or 45.1 "cyt b" °C, 1 min (extended) at 72°C 35 cycles, followed by a final extension of 72°C for 5 min. PCR products were run on a 1.5% agarose gel containing ethidium bromide and visualized under UV light.

Sequencing: PCR products were purified using a QIAquick PCR Product extraction kit (Qiagen Inc.) according to manufacturer’s recommendations. A purified PCR product was sequenced in the forward and/or reverse directions; direct sequencing of the resulted product was done by using same primer sets used for amplification with an automated DNA sequencer (Applied Biosystems 3130 automated DNA Sequencer ABI, 3130, USA). Using a ready reaction Bigdye Terminator V3.1 cycle sequencing kit. (Perkin-Elmer/Applied Biosystems, Foster City, CA).

Phylogenetic analysis: The newly obtained sequences of P. ambiguus were checked manually. Also, A BLAST^® analysis (Basic Local Alignment Search Tool) was initially performed to establish sequence identity to GenBank accessions. Nucleotide sequences were compared with GenBank sequences using the BLAST system and translated into amino acid sequences. Sequences, included the newly obtained one were aligned using MEGA7 software and ambiguously analyzed in comparison with gene sequence of P. ambiguus and other oxyurids.

Statistical analysis: When required, statistical analysis performed using Microsoft Excel software for Windows.

RESULTS

DNA from P. ambiguus was positive by PCR using the specific primer at length of a partial fragment of cox1 mtDNA was 750 bp and partial fragments of cyt b mtDNA fragment was 700 bp in length (Fig. 1).

The partial gene sequences obtained from P. ambiguus samples from Egyptian domestic rabbits were analysed using MEGA 7 software in comparison with other peers of the accession numbers (MG991568 for cox1 1 gene) and (MH025840 for cyt b gene) showing that the sequence was similar to each other (similarity 99% for cox1 and 98% for cyt b).

To make phylogenetic relationships of Egyptian P. ambiguus, the amino acid sequences conceptually translated from individual genes of the mt genes of P. ambiguus were done. The Neighbour-Joining (NJ) analysis was carried out based on Kimura 2-parameter with 1500 replicates (Fig. 2a, b, respectively), illustrated GenBank sequences of P. ambiguus (KF472054.1, KF472055.1, KF472059.1, KF472062.1) and KT875315.1 belonged to Rauschtineria eutamii. From this tree showed two branches, first cluster included the present sequence (MG 991568), KF472062.1 and KF472059.1 had very strong support of 100%. In contrast, the Fig. 2b shown the Neighbour-Joining (NJ) analysis of cyt b and a strongly supported cluster with a confidence level of 100% was observed between the sequence from this study with other sequences from GenBank.

DISCUSSION

Molecular biology tools have contributed effectively in association with traditional morphological techniques to identify, differentiate between closely related species as well as draw the relationships and evolutionary history of parasites¹⁶. Most important morphological features of Passalurus spp. includes the characteristic shape of the oesophagus, male genital area and female posterior end. But, based solely on ordinary morphological examination; it is difficult to differentiate in-between two closely-related pinworms namely "P. ambiguus" and "P. nonanulatus"^17,18. In a previous study by Sultan et al.⁸, we confirm the Egyptian isolates of Passalurus sp., as "P. ambiguus" by ordinary and electron microscope examination.

In this study, PCR was employed for amplification of partial specific mtDNA genes (cox1 and cyt b), for confirmation of the species and draw its relationships with other oxyurids. We used cox1 gene as, it is the more conserved gene within oxyurids¹⁹. Our phylogeny study comes in line with that obtained by Liu et al.¹⁵, in that P. ambiguus is much similar to Wellcomia siamensis than to Enterobius vermicularis.

Fig. 1(a-b):

Agarose gel showing PCR product for Passalurus ambiguus, (a) cox1, lane 1 shows negative control, lanes 2, 3 shows positive reaction at 750 bp and (b) cyt b, lanes 2, 3 shows positive reaction at 700 bp M: Marker

Fig. 2(a-b):

Neighbour joining phylogenetic tree of P. ambiguus from Egyptian domestic rabbits of (a) cox1 gene with 1500 bootstrap replications and (b) ctyb gene with 1500 bootstrap replications Genbank accession numbers of sequences were used to generate the tree are used. KF472062, KF472059, KF472055 and KF472054 are for P. ambiguous isolates form other parts of the world. Rauschtineria eutamii with accession number KT875315 was used as outside group. KF472078, KF472088, KF472076, KF472074 and KF472079 are for P. ambiguous isolates form other parts of the world. Labiostomum (Eugenuris) sp. with accession number KY905885 was used as outside group

Fewer mt genome data are available for Oxyuridomorpha "i.e., oxyurids", which made it very difficult to estimate the phylogenetic relationships until, the complete mt genomes of some pinworms i.e., E. vermicularis, W. siamensis, Oxyuris equi and P. ambiguus were sequenced by Liu et al.¹⁵ and Zhang et al.¹⁹ respectively; the results of all previous studies are in-line with ours in that O. equi, E. vermicularis, P. ambiguus and W. siamensis are closely related species. Studying and analysis of pinworms mtDNA genes showed to be a powerful tool in identification, studying evolutionary history, geographical variations among same species and population differences^20-22, our results are in the same way. Further studies including more genera and species of oxyurids from different hosts and regions may help in better understanding of the relationships and evolutionary history of these parasites.

CONCLUSION

In conclusion, rabbit pinworms isolate from Egypt are much genetically similar to those from the rest of the world, also mt DNA genes proved to be good genetic targets to identify and study pinworms of medical and veterinary importance. Further studies on pinworms from man and animals morphology and genetics are necessary.

SIGNIFICANCE STATEMENT

Pinworms are an important group of helminths of both medical and veterinary importance. Passalurus ambiguus is one the most common pinworms, infecting rabbits and may cause economic losses. Therefore, identification of helminth species via molecular tools becomes important to address its relationships and evolutionary history. Results of this study demonstrates the suitability and need of genetic markers such cox1 and cyt b in identification of pinworms.

ACKNOWLEDGMENT

Authors wish to express their thanks to all members of Parasitology Department, Faculty of Veterinary Medicine, Kafrelsheikh University for their sincere help during this study.