Phylogenetic analysis of Spirocerca lupi and Spirocerca vulpis reveal high genetic diversity and intra-individual variation

Background: Spirocerca 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. Results: A 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. Conclusions: These 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