A real-time PCR tool for the surveillance of zoonotic Onchocerca lupi in dogs, cats and potential vectors

Abstract

The ocular onchocercosis is caused by the zoonotic parasite Onchocerca lupi (Spirurida: Onchocercidae). A major hindrance to scientific progress is the absence of a reliable diagnostic test in affected individuals. Microscopic examination of skin snip sediments and the identification of adults embedded in ocular nodules are seldom performed and labour-intensive. A quantitative real-time PCR (qPCR) assay was herein standardized for the detection of O. lupi DNA and the results compared with microscopic examination and conventional PCR (cPCR). The specificity of qPCR and cPCR was assessed by processing the most common filarial nematodes infecting dogs, skin samples from O. lupi infected (n = 35 dogs) or uninfected animals (n = 21 dogs; n = 152 cats) and specimens of potential insect vector (n = 93 blackflies; n = 59 mosquitoes/midges). The analytical sensitivity of both assays was assessed using 10-fold serial dilutions of DNA from adult specimen and from a pool of microfilariae. The qPCR on skin samples revealed an analytical specificity of 100% and a sensitivity up to 8 x 10−1 fg/2μl O. lupi adult-DNA and up to 3.6 x 10−1 pg/2μl of mfs-DNA (corresponding to 1 x 10−2 mfs/2μl). Only 9.5% O. lupi-infected skin samples were positive for cPCR with a sensitivity of 8 x 10−1 pg/2μl of DNA. Out of 152 blackflies and mosquitoes/midges, eight specimens experimentally infected (n = 1 S. erythrocephalum; n = 1 S. ornatum; n = 6 Simulium sp.) were positive by qPCR. The qPCR assay herein standardized represents an important step forward in the diagnosis of zoonotic onchocercosis caused by O. lupi, especially for the detection and quantification of low number of mfs. This assay provides a fundamental contribution for the establishment of surveillance strategies aiming at assessing the presence of O. lupi in carnivores and in insect species acting as potential intermediate hosts. The O. lupi qPCR assay will enable disease progress monitoring as well as the diagnosis of apparently clinical healthy dogs and cats.

Major antigen and paramyosin proteins as candidate biomarkers for serodiagnosis of canine infection by zoonotic Onchocerca lupi

“…However, the immunological potential of linear antigenic epitopes has been demonstrated by microarray screening of the entire proteome of different pathogens [61][62][63], as it is the case of O. volvulus for which three peptide motifs with an IgG1, IgG3, IgE and IgM response profile were identified [64]. Furthermore, though Paramyosin is a widely conserved protein across species with well-known antigenic potential [65,66], the immunological properties of Ol-PARA [25] have been herein investigated by the identification of the three linear epitopes (LEEARRRLE, SRLQSEVEVLIVDL, MQVDEEHKMF), which showed a high intensity of reactivity when tested against serum sample of O. lupi infected dog.…”

“…However, the results of these procedures may be (false-) negative in the case of infections with immature or not reproducing worms and according to the day-time of the sampling, considering the circadian rhythm of mfs [8]. Even if, PCR-based DNA assays have been developed [7,25] and preliminary investigations on serology have been attempted [26,27], a serological, non-invasive diagnosis is still missing. Although, the preliminary immunological properties of paramyosin protein of O. lupi (Ol-PARA) has been previously evaluated, no immunoreactive peptides have been identified [27].…”

Development of a multiplex qPCR-based approach for the diagnosis of Dirofilaria immitis, D. repens and Acanthocheilonema reconditum

“…Acanthocheilonema reconditum, A. dracunculoides, Cercopithifilaria grassii, Brugia ceylonensis, B. patei, B. malayi, B. pahangi, Onchocerca lupi and Thelazia callipaeda [1][2][3][4]. These arthropod-borne filarioids produce blood, cutaneous or mucous microfilariae, where they are available to arthropod vectors [5].…”

First report ofOnchocerca lupifrom Israel and confirmation of two genotypes circulating among canine, feline and human hosts

“…Molecular assays have been employed to detect O. lupi in dogs, humans, cats and black flies by targeting three mitochondrial loci: the cytochrome oxidase c subunit 1 ( cox 1) (Hassan et al ., 2015; Maia et al ., 2015; Otranto et al ., 2015; Hodzic et al ., 2018; Latrofa et al ., 2018), the 12S rRNA (Faisca et al ., 2010; Mowlavi et al ., 2014) and the reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 5 ( nad 5) (Labelle et al ., 2013; McLean et al ., 2017). Mitochondrial markers are ideal for phylogenetic and epidemiologic studies since they are clonal, they rarely or never undergo recombination, are mostly of maternal inheritance and evolve faster than nuclear genes (Le et al ., 2000).…”