Liu et al. Parasites & Vectors (2015) 8:100 DOI 10.1186/s13071-015-0697-5 RESEARCH Open Access The complete mitochondrial genome of the gullet worm Gongylonema pulchrum: gene content, arrangement, composition and phylogenetic implications Guo-Hua Liu1†, Yan-Qing Jia2†, Ya-Nan Wang2, Guang-Hui Zhao2* and Xing-Quan Zhu1* Abstract Background: Gongylonema pulchrum (Nematoda: Gongylonematidae), a thread-like spirurid gullet worm, infects a range of mammalian definitive hosts, including cattle, pigs, equines, goats, primates and humans, and can cause gongylonemiasis. Methods: In the present study, the complete mitochondrial (mt) genome of G. pulchrum was obtained using Long-range PCR and subsequent primer walking. The phylogenetic position of G. pulchrum within the Spiruromorpha was established using Bayesian analyses of the protein-coding genes at the amino acid level. Results: The length of this AT-rich (75.94%) mt genome is 13,798 bp. It contains 12 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes and one non-coding region. The gene arrangement is the same as those of Thelazia callipaeda (Thelaziidae) and Setaria digitata (Onchocercidae), but distinct from that of Heliconema longissimum (Physalopteridae). Phylogenetic analyses, based on the concatenated amino acid sequence data for all 12 protein-coding genes using Bayesian inference (BI) method, showed that G. pulchrum (Gongylonematidae) was more closely related to Spirocerca lupi (Spiruroidea) than other members of the infraorder Spiruromorpha. Conclusions: The present study represents the first mt genome sequence for the family Gongylonematidae, which provides the opportunity to develop novel genetic markers for studies of epidemiology, population genetics and systematics of this nematode of human and animal health significance. Keywords: Gongylonema pulchrum, Gongylonemiasis, Mitochondrial genome, Phylogenetic analyses Background animal hosts, including bears, camels, cattle, cervids, Gongylonema pulchrum Molin, 1857, known as the donkeys, equines, goats, non-human primates, pigs, and ‘gullet worm’ because of its location in the upper digestive sheep, causing major economic losses [2]. Fortunately, gon- system of its definitive hosts, has a worldwide distribution gylonemiasis can be treated effectively using anthelmintics, and is responsible for gongylonemiasis of humans, some- such as levamisole, mebendazole and ivermectin [3]. times causing serious complaints, e.g., expectoration of Human gongylonemiasis, a neglected parasitic disease, blood, numbness of tongue, vomiting, pharyngitis and sto- has been reported from many countries (e.g., Australia, matitis [1]. G. pulchrum also infects domestic and wild Bulgaria, Ceylon, China, France, Germany, Hungary, Iran, Japan, Laos, Morocco, New Zealand, Soviet Union, Spain, Sri Lanka, Turkey and USA). Recently, human gongylone- * Correspondence: [email protected]; [email protected] †Equal contributors miasis has frequently been reported from China [4], France 2College of Veterinary Medicine, Northwest A&F University, Shaanxi Province, [5,6] and USA [7]. Clearly, the increased frequency and Yangling 712100, Peoples Republic of China more widespread occurrence of clinical cases of human 1State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research gongylonemiasis mean that knowledge on enhanced diag- Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province nosis, treatment and control is needed. Although molecular 730046, Peoples Republic of China © 2015 Liu et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu et al. Parasites & Vectors (2015) 8:100 Page 2 of 8 markers in portion of mitochondrial (mt) DNA and the in- cox1, cox2 and cox3 regions of nematodes within order ternal transcribed spacer (ITS) regions of nuclear ribosomal Spirurata (Figure 1), three overlapping amplicons of the DNA (rDNA), have found utility for taxonomic and epi- complete mt genome were amplified by Long-PCR [21]. demiological studies of G. pulchrum [8], there is still a pau- PCRs were conducted in 25 μl reaction volumes contain- city of information on G. pulchrum in different hosts and ing 2 mM MgCl2, 0.2 mM each of dNTPs, 2.5 μl 10× countries around the world. Taq buffer, 2.5 μM of each primer and 0.5 μlLATaq Due to its maternal inheritance, fast rate of evolutionary DNA polymerase (5 U/μl, TaKaRa). PCR cycling condi- change, lack of recombination and relatively conserved tions were as follows: 92°C for 2 min (initial denatur- genome structures [9], mt genomes have been widely used ation), then 92°C for 10 s (denaturation), 45°C for 30 s as genetic markers for population genetic structure and (annealing), and 60°C for 8 min (extension) for 9 cycles, the study of phylogenetic relationships among nematodes followed by 92°C for 10 s, 45°C for 30 s (annealing), and and trematodes [10]. In particular, concatenated amino 60°C for 9 min (extension) for 25 cycles, with a cycle acid sequences, derived from the protein-coding genes, elongation of 10 s for each cycle and a final extension at lend themselves for assessing systematic relationships of 60°C for 10 min. No-template and known-positive con- parasitic nematodes [11-19]. The objectives of the present trols were included in each run. Amplicons were study were to characterize the mt genome of G. pulchrum, column-purified using TIANgel Midi Purification Kit the first representative of the family Gongylonematidae, (TIANGEN, Beijing, China). Following an electrophor- and to assess the phylogenetic position of this zoonotic etic analysis of quality, purified amplicons were se- nematode in relation to other nematodes of the infraorder quenced using a primer walking strategy [21] with Spiruromorpha. primers listed in Additional file 1 by Invitrogen Com- pany (Shanghai, China). Methods Ethics statement Sequence analyses The performance of this study was strictly according to Sequences were assembled manually and aligned against the recommendations of the Guide for the Care and Use the complete mt genome sequences of Spirocerca lupi of Laboratory Animals of the Ministry of Health, China, [22] using the computer program MAFFT 7.122 [23] to and our protocol was reviewed and approved by the Re- identify gene boundaries. Each gene was translated into search Ethics Committee of Northwest A&F University. its amino acid sequence using the invertebrate mito- chondrial genetic code in MEGA 5 [24]. The translation Parasite collection initiation and termination codons were identified to Adult specimens of G. pulchrum were collected from avoid gene overlap and to optimize the similarity be- the oesophagus of a naturally infected goat in Shenmu, tween the gene lengths of closely related species of the Shaanxi province, China, with no specific permits being infraorder Spiruromorpha. The program tRNAscan-SE required by the authority for the sample collection. [25] was used to find tRNA and infer their secondary structure, putative secondary structures of 19 tRNA genes Genomic DNA extraction were identified, and the remaining three tRNA genes The gullet worms were washed extensively in physio- (tRNA-Arg, tRNA-Ser AGN and tRNA-Ser UCN)werein- logical saline, identified morphologically to species ac- ferred by recognizing potential secondary structures and cording to existing keys and descriptions [20], fixed in anticodon sequences by eye [26]. Two rRNA genes were ethanol and then stored at −20°C until use. The mid- predicted by comparison with those of closely related body section of each worm was used for the isolation of nematodes of the infraorder Spiruromorpha [15,22]. total genomic DNA using proteinase K digestion and mini-column purification (TIANamp Genomic DNA Phylogenetic analyses Purification System, TIANGEN). The identity of the spe- Amino acid sequences inferred from published mt ge- cimen was verified by sequencing regions of the ITS-1 nomes representing 12 species of the infraorder Spiru- and ITS-2 rDNA using an established method [8]; the romorpha (Brugia malayi: GenBank accession no. two regions were 99.7% and 100% identical to previously NC_004298; Wuchereria bancrofti: JN367461; Chandle- published sequences for G. pulchrum from Bos taurus in rella quiscali: NC_014486; Loa loa: NC_016199; Acantho- Iran (GenBank accession nos. AB495392 and AB513721, cheilonema viteae: NC_016197; Onchocerca flexuosa: respectively). NC_016172; Onchocerca volvulus: AF015193; Dirofilaria immitis: NC_005305; Setaria digitata: NC_014282; S. lupi: Long-PCR and sequencing KC305876; Thelazia callipaeda: JX069968; Heliconema Using three pairs of specific primers (Table 1) designed longissimum: NC_016127) were included in the present according to relatively conserved regions within the analysis, using Toxascaris leonina (NC_023504) as the Liu et al. Parasites & Vectors (2015) 8:100 Page 3 of 8 Table 1 Primers used to amplify the mitochondrial genome of Gongylonema pulchrum in three overlapping long PCR fragments Name of primer Sequence (5′ to 3′) Extension positions Product lengths