Mitochondrial cytochrome c oxidase subunit 1 (cox1) gene sequence of Spirocerca lupi (Nematoda, Spirurida): Avenues for potential implications

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Abstract

Canine spirocercosis is a life-threatening parasitosis caused by Spirocerca lupi (Nematoda, Spirurida) that is presently emerging in several countries. This study characterised an informative region within the mitochondrial (mtDNA) gene encoding for the cytochrome c oxidase subunit 1 (cox1) of S. lupi by Polymerase Chain Reaction (PCR)-coupled sequencing. Specimens from five different countries in Europe, Asia and Africa were examined and two different sequence variants of cox1 (i.e. haplotypes) were determined, displaying nucleotidic variation at 6 of 689 positions. All of these positions were invariable among all the parasite individuals from Europe (haplotype 1) and among the African and Asian individuals (haplotype 2), but differed between Europe and Asia/Africa. The S. lupi cox1 sequences were consistent with those of other common Spirurida previously reported at both nucleotidic and phylogenetic levels. This study provides molecular information essential for identification of the nematode, irrespective of its life cycle stage. Crucial implications for the specific molecular diagnosis of clinical spirocercosis and investigation of the evolution, population genetics, ecology and epidemiology of S. lupi are discussed.

Introduction

Spirocerca lupi (Nematoda, Spirurida) adults primarily live in oesophageal mural nodules, consisting of a fibrous, tumour-like tissue in their hosts, i.e. dogs and other carnivores. Its life cycle is indirect, and animals become infected by ingesting an intermediate (i.e. coprophagous beetles) or a paratenic (e.g. birds, lizards or rodents) host (Bailey, 1972, Anderson, 2000). The disease is characterised by several symptoms of varying severity, including anaemia, vomiting/regurgitation, melena, vertebral spondylitis, aortic aneurysms and dyspnoea due to aspiration pneumonia and pyothorax. It may be life-threatening due to aortic aneurism rupture. Moreover, the oesophageal nodules may go through malignant transformation to fibrosarcoma and osteosarcoma and metastasize to the lungs (Bailey, 1972, Ivoghli, 1977, Johnson, 1992, Mazaki-Tovi et al., 2002, Ranen et al., 2004, Klainbart et al., 2007).

Canine spirocercosis has been widely reported in North and South America (Georgi et al., 1980, Melendez and Suarez-Pellin, 2001, Santos et al., 2004), the Middle East (Mazaki-Tovi et al., 2002, Ranen et al., 2004), Africa (Brodey et al., 1977, Lobetti, 2000, Joubert et al., 2005) and Europe (Bordeau, 1986, Kramer et al., 2003, Mylonakis et al., 2006), and has been described as an emerging disease, with a significant increase in the annual number of cases in several regions over the past decade (Lobetti, 2000, Reche-Emonot et al., 2001, Mazaki-Tovi et al., 2002, Oliveira-Sequeira et al., 2002, Kramer et al., 2003, Ranen et al., 2004, Santos et al., 2004, Joubert et al., 2005, Mylonakis et al., 2006; Traversa, unpublished cases).

Despite its diffusion and importance in canine medicine, comprehensive information on the epidemiology of S. lupi infections is lacking in many areas, partly due to difficulties in the diagnosis in living animals and unapparent subclinical infections (Mazaki-Tovi et al., 2002). The clinical signs of spirocercosis may be confused with those of other diseases and the parasitological diagnosis shows inherent limits. Moreover, the typical lesions caused by S. lupi are oesophageal nodules that can potentially progress to neoplastic transformation, and these are usually diagnosed through oesophagoscopy and radiography, that are invasive and may be insensitive, respectively. Both modalities may not allow an aetiological diagnosis.

Although canine spirocercosis is an emerging disease, and many studies on its epidemiology, clinical signs and treatment (Mazaki-Tovi et al., 2002, Ranen et al., 2004) as well as molecular investigations of several parasites of several orders have been recently published (e.g. Zhu et al., 2001, Chilton, 2004, Pozio and Murrell, 2006), no molecular information of S. lupi is available, with exception of partial ribosomal DNA sequences registered in GenBank™ (Accession numbers AY751500 and AY751497).

Regions within the mitochondrial DNA (mtDNA) have recently been proven useful in characterisation of population genetics, biology, epidemiology and diagnosis of several parasitic nematodes of human and veterinary importance (Hu et al., 2004, Hu and Gasser, 2005). DNA sequencing of informative regions within the mtDNA gene encoding for the cytochrome c oxidase subunit 1 (cox1) revealed data for basic and applied potential studies for Spirurida nematodes (Casiraghi et al., 2001, Ngarmamonpirat et al., 2005, Otranto et al., 2005, Ando et al., 2006). In particular, the cox1 gene is useful for studies on the sequence variability and population genetics of parasitic nematodes and arthropods (Blouin, 2002, Hu et al., 2004, Hu and Gasser, 2005, Shao and Barker, 2006), as well as for diagnostic purposes by using techniques able to overcome inherent limits of the classical approaches (Caldeira et al., 2003, Traversa and Otranto, 2006). Hence, we considered the S. lupi cox1 promising for the molecular investigation of this parasite and its molecular characterisation, and to provide a crucial step for further studies of canine spirocercosis. The aims of the present work were to characterise, for the first time, the sequence of the S. lupi cox1 gene, to promote basic knowledge on the mtDNA of Spirurida nematodes, to assess the sequence variation level within S. lupi isolates from different geographical areas, and to discuss the potential benefits of such molecular information as a foundation for ecological, epidemiological and diagnostic application to canine spirocercosis.

Section snippets

Parasite materials

Specimens of S. lupi were collected from naturally infected dogs from different countries where the parasite is endemic or presently emerging, i.e. Israel (n = 7), Iran (n = 3), South Africa (n = 4), Italy (n = 5) and Austria (n = 1). After morphological speciation, the nematodes were washed extensively in physiological saline and fixed in absolute ethanol pending molecular analysis.

DNA extraction and Polymerase Chain Reaction

Individual nematodes were exposed to liquid nitrogen and then subjected to the genomic DNA extraction using a commercial

Results

All 20 cox1 PCR products representing S. lupi from dogs from different countries were of the expected length (i.e. 689 bp), with no insertions and deletions (i.e. indels) detected in any of the sequences, and an A + T content of 64%. Two different types of cox1 sequence (i.e. haplotypes) were detected in all the 20 sequences determined. In particular, no nucleotide variation was detected within specimens from Europe (Italy and Austria) (haplotype 1), and within specimens from Asia (Israel and

Discussion

The present work reports the first informative molecular characterisation of a mtDNA region of the parasitic nematode S. lupi, expands the knowledge on Spiruridal mtDNA, and suggests that the S. lupi cox1 gene is a promising genetic target for a holistic approach to the study of canine spirocercosis. However, it was not the purpose of the present work to perform genetic population studies of S. lupi or to produce detailed phylogeny for this important parasite. The main aim of the present work

Acknowledgements

The authors are grateful to Joop Boomker (University of Pretoria, South Africa), Soraya Naem (University of Urmia, Iran) and Sabine Brandt (University of Wien, Austria) who provided specimens of S. lupi.

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