Genetic evidence for the presence of two species ofOnchocercafrom the wild boar in Japan

Abstract

Summary :In order to clarify the genetic differences between Onchocerca dewittei japonica, the causative agent of zoonotic onchocerciasis in Japan and a related undescribed Onchocerca sp., both parasitizing wild boar (Sus scrofa) of which the infective larval stages are indistinguishable from each other, we compared the sequences of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene region from four infective larvae (recovered from experimentally infected black flies), one microfilaria, and one adult of O. dewittei japonica, and from one infective larva (recovered from an experimentally infected black fly), one microfilaria, and a pool of several microfilariae of O. sp. The length of the CO1 gene region was 649 bp for all samples but there was a difference of 8.8 to 9.4 % in the sequences between the two species although there were intraspecific variations of 0 to 0.5 %.

Blackfly vectors of zoonotic onchocerciasis in Japan

“…sensu Fukuda et al, 2008, another parasite of wild boar Onchocerca sp. sensu Fukuda et al, 2008, another species from wild boar, was first distinguished at the microfilarial stage by its body length, which was much greater than that of O. dewittei japonica, and its distinctness as a species was confirmed by DNA analysis (Fukuda et al, 2010b). The infective larvae of Onchocerca sp.…”

Molecular identification of infective larvae of three species ofOnchocercafound in wild-caught females ofSimulium bidentatumin Japan

“…The mitochondrial CO1 gene was amplified using the primer set CO1intF-CO1intR (Casiraghi et al, 2001) as described elsewhere (Fukuda et al, 2010). Briefly, PCR was performed in a final volume of 50 µl containing 1 × Ex Taq buffer including 2 mM Mg 2+ , 200 µM each of dNTPs, 0.1 µM each of primers, 1.25 units of Ex Taq Polymerase (TAKARA BIO INC., Otsu, Japan), and 3 µl of template DNA under the following thermal conditions: an initial denaturation at 94°C for 3 min, followed by five cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min and 37 cycles of 94°C for 30 s, 48°C for 30 s, and 72°C for 1 min.…”

“…(nucleotide difference: 0-0.2 %), where differences between type B and other Onchocerca species were 7.5-11.8 %. The nucleotide and amino acid sequences of three larvae of type A (L-7-L-9) had difference of more than 9.1 % and 0.9 % from any Oncho- (Fukuda et al, 2010). b The CO1 sequence was identical to that of AB518694 (Fukuda et al, 2010).…”

“…The nucleotide and amino acid sequences of three larvae of type A (L-7-L-9) had difference of more than 9.1 % and 0.9 % from any Oncho- (Fukuda et al, 2010). b The CO1 sequence was identical to that of AB518694 (Fukuda et al, 2010). c The CO1 sequence was identical to that of O. sp.…”

“…from wild boar (abbreviated O. sp. hereafter) and O. gutturosa Neumann, 1910 from cattle (Bos taurus Linnaeus, 1758), all of which are morphologically indistinguishable from one another at the infective larval stage (Fukuda et al, 2010). To search for the natural vectors of O. dewittei japonica, we investigated infections of black flies with filarial larvae in Oita, Japan and attempted to identify infective larvae found on the basis of the analysis of the mitochondrial CO1 and 12S ribosomal RNA (12S rRNA) gene sequences.…”