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Detection and quantification of Spirocerca lupi by HRM qPCR in fecal samples from dogs with spirocercosis.

ABSTRACT: Spirocerca lupi, the dog oesophageal nematode, causes a potentially fatal disease in domestic dogs, and is currently clinically diagnosed by coproscopy and oesophagoscopy. To date, a single molecular method, a semi-nested PCR, targeting the cox1 gene, has been developed to aid in the diagnosis of spirocercosis. The present study describes three novel high-resolution melt (HRM) quantitative PCR (qPCR) assays targeting fragments of the ITS1, 18S and cytb loci of S. lupi. The performance of these molecular assays in feces was compared to fecal flotation and to the previously described cox1 gene semi-nested PCR in 18 fecal samples from dogs with clinical oesophageal spirocercosis diagnosed by oesophagoscopy.The HRM qPCR for ITS1 and 18S were both able to detect 0.2 S. lupi eggs per gram (epg), while the HRM qPCR for the cytb and the semi-nested PCR for the cox1 detected 6 epg and 526 epg, respectively. Spirocerca lupi was detected in 61.1%, 44.4%, 27.8%, 11.1% and 5.6% of the fecal samples of dogs diagnosed with spirocercosis by using the ITS1 and 18S HRM qPCR assays, fecal flotation, cytb HRM qPCR and cox1 semi-nested PCR, respectively. All dogs positive by fecal flotation were also positive by ITS1 and 18S HRM qPCRs. Quantification of S. lupi eggs was successfully achieved in the HRM qPCRs and compared to the fecal flotation with no significant difference in the calculated concentrations between the HRM qPCRs that detected the 18S and ITS1 loci and the fecal flotation. The HRM qPCR for the 18S cross-amplified DNA from Toxocara canis and Toxascaris leonina. In contrast, the HRM qPCR for ITS1 did not cross-amplify DNA from other canine gastrointestinal parasites.This study presents two new molecular assays with significantly increased sensitivity for confirming and quantifying fecal S. lupi eggs. Of these, the HRM qPCR for ITS1 showed the best performance in terms of the limit of detection and absence of cross-amplification with other parasites. These assays will be useful in detecting infection and for follow-up during therapy.

SUBMITTER: Rojas A

PROVIDER: S-EPMC5606040 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Detection and quantification of Spirocerca lupi by HRM qPCR in fecal samples from dogs with spirocercosis.

Rojas Alicia A Segev Gilad G Markovics Alex A Aroch Itamar I Baneth Gad G

Parasites & vectors 20170919 1

<h4>Background</h4>Spirocerca lupi, the dog oesophageal nematode, causes a potentially fatal disease in domestic dogs, and is currently clinically diagnosed by coproscopy and oesophagoscopy. To date, a single molecular method, a semi-nested PCR, targeting the cox1 gene, has been developed to aid in the diagnosis of spirocercosis. The present study describes three novel high-resolution melt (HRM) quantitative PCR (qPCR) assays targeting fragments of the ITS1, 18S and cytb loci of S. lupi. The per ...[more]

PMID: 28927435

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Project description:BackgroundSpirocerca lupi is a parasitic nematode of canids that can lead to a severe and potentially fatal disease. Recently, a new species, Spirocerca vulpis, was described from red foxes in Europe, suggesting a high genetic diversity of the Spirocerca spp. infecting canids. The genetic variation and phylogenetic relationships of S. lupi collected from naturally-infected domestic dogs from Australia, Hungary, Israel, Italy, India and South Africa, and S. vulpis from red foxes from Bosnia and Herzegovina, Italy and Spain, was studied using mitochondrial and rDNA markers.ResultsA high intra-individual variation was found in the first internal transcribed spacer (ITS1) locus in all Spirocerca spp., ranging between 0.37-2.84%, with up to six haplotypes per specimen. In addition, a combination of phylogenetic and haplotype analyses revealed a large variability between S. lupi specimens collected from different geographical locations using the ITS1 (0.37-9.33%) and the cytochrome c oxidase subunit 1 (cox1) gene (1.42-6.74%). This genetic diversity led to the identification of two S. lupi genotypes circulating among dogs (PTP support > 0.829), including genotype 1 found in S. lupi from Australia, India, Israel and South Africa, and genotype 2 represented by specimens from Hungary and Italy. These genotypes presented pairwise nucleotide distances of 0.14%, 8.06% and 6.48 ± 0.28% in the small rDNA subunit (18S), ITS1 and cox1 loci, respectively. Additionally, Nei's genetic distance in the ITS1 showed a further subdivision of genotype 1 worms into 1A (Israel and South Africa) and 1B (Australia and India). A morphological analysis of the anterior and posterior extremities of genotype 1 and genotype 2 worms using scanning electron microscopy did not show any differences between the specimens, contrary to the morphological differences between S. lupi and S. vulpis.ConclusionsThese findings demonstrate the high genetic variability among Spirocerca spp. from different geographical locations, thereby expanding our understanding of the epidemiology, evolution and phylogenetic variability within the genus.

Dataset Information

Detection and quantification of Spirocerca lupi by HRM qPCR in fecal samples from dogs with spirocercosis.

Publications

Detection and quantification of Spirocerca lupi by HRM qPCR in fecal samples from dogs with spirocercosis.

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