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

ABSTRACT: Onchocerca lupi (Spirurida: Onchocercidae) is a filarial worm parasitizing domestic carnivores and humans. Adult nematodes usually localize beneath in the sclera or in the ocular retrobulbar of infected animals, whilst microfilariae are found in the skin. Therefore, diagnosis of O. lupi is achieved by microscopic and/or molecular detection of microfilariae from skin biopsy and/or surgical removal of adults from ocular tissues of infected hosts. An urgent non-invasive diagnostic tool for the diagnosis of O. lupi in dog is mandatory. In this study, an immunoproteomic analyses was performed using a combination of immunoblotting and mass spectrometry techniques. Onchocerca lupi major antigen (Ol-MJA) and paramyosin (Ol-PARA) proteins were identified as potential biomarkers for serodiagnosis. Linear epitopes were herein scanned for both proteins using high-density peptide microarray. Sera collected from dog infected with O. lupi and healthy animal controls led to the identification of 11 immunodominant antigenic peptides (n = 7 for Ol-MJA; n = 4 for Ol-PARA). These peptides were validated using sera of dogs uniquely infected with the most important filarioids infesting dogs either zoonotic (Dirofilaria repens, Dirofilaria immitis) or not (Acanthocheilonema reconditum and Cercopithifilaria bainae). Overall, six antigenic peptides, three for Ol-MJA and for Ol-PARA, respectively, were selected as potential antigens for the serological detection of canine O. lupi infection. The molecular and proteomic dataset herein reported should provide a useful resource for studies on O. lupi toward supporting the development of new interventions (drugs, vaccines and diagnostics) against canine onchocercosis.

SUBMITTER: Latrofa MS

PROVIDER: S-EPMC7875354 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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

Latrofa Maria Stefania MS Palmisano Giuseppe G Annoscia Giada G Pierri Ciro Leonardo CL Chandrashekar Ramaswamy R Otranto Domenico D

PLoS neglected tropical diseases 20210210 2

Onchocerca lupi (Spirurida: Onchocercidae) is a filarial worm parasitizing domestic carnivores and humans. Adult nematodes usually localize beneath in the sclera or in the ocular retrobulbar of infected animals, whilst microfilariae are found in the skin. Therefore, diagnosis of O. lupi is achieved by microscopic and/or molecular detection of microfilariae from skin biopsy and/or surgical removal of adults from ocular tissues of infected hosts. An urgent non-invasive diagnostic tool for the diag ...[more]

PMID: 33566799

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Project description:BackgroundOf increasing importance to the medical and veterinary communities is the zoonotic filarioid nematode Onchocerca lupi. Onchocercosis, thus far found in wolves, dogs, cats and humans, is diagnosed via skin snips to detect microfilariae and surgical removal of adults from the eye of the host. These methods are time-consuming, laborious and invasive, highlighting the need for new tools for the diagnosis of O. lupi in susceptible hosts. Symptoms related to the presence of the adults in the eye can range from none apparent to severe, including blindness. No reliable chemotherapeutic protocols are available, as yet, to eliminate the infection. Paramyosin, an invertebrate-specific protein, has been well-studied as an allergen, diagnostic marker and vaccine candidate. The aim of this study, therefore, was to isolate and characterise paramyosin from O. lupi to assess its suitability for the development of a serological diagnostic assay.MethodsThe adult and microfilarial stages of O. lupi were isolated from the eyes and skin of a 3-year-old male dog. Total RNA was extracted and reverse transcribed into single stranded cDNA. Reverse-transcription PCR was used to isolate a full-length paramyosin cDNA from adult worms and to investigate the temporal expression patterns of this gene. All amplicons were sequenced using dideoxy chain termination sequencing. Bioinformatics was used to predict the amino acid sequence of the gene, to compare the DNA and protein sequences with those available in public databases and to investigate the phylogenetic relationship of all molecules. Antibody binding sites were predicted using bioinformatics and mapped along with published antigenic epitopes against the O. lupi paramyosin protein. The native protein, and three smaller recombinantly expressed peptides, were subjected to western blot using serum from dogs both positive and negative for O. lupi.ResultsParamyosin of O. lupi was herein molecularly characterized, encoded by a transcript of 2,643 bp and producing a protein of 881 amino acids (101.24 kDa). The paramyosin transcript was detected, by reverse transcription PCR, in adults and microfilariae, but not in eggs. Phylogenetic analysis indicates that this molecule clusters with paramyosins from other filarioids to the exclusion of those from other taxa. A total of 621 unique antibody binding epitopes were predicted for this protein and another 28 were conserved in other organisms. This information was used to design three peptides, for recombinant expression, to identify the antibody binding epitope(s) and reduce potential cross-reactivity with serum from dogs infected with other filarioid nematodes. Native paramyosin, purified from microfilariae and adults, was detected by antibodies present in serum from dogs with known O. lupi infections.ConclusionsData provided herein may assist in the development of a serological diagnostic test, based on antibodies to O. lupi paramyosin, for the diagnosis of this infection, in order to gain more information on the real distribution of this little known filarioid of zoonotic concern.

Project description:The genus Onchocerca encompasses parasitic nematodes including Onchocerca volvulus, causative agent of river blindness in humans, and the zoonotic Onchocerca lupi infecting dogs and cats. In dogs, O. lupi adult worms cause ocular lesions of various degrees while humans may bear the brunt of zoonotic onchocercosis with patients requiring neurosurgical intervention because of central nervous system localization of nematodes. Though the zoonotic potential of O. lupi has been well recognized from human cases in Europe, the United States and the Middle East, a proper therapy for curing this parasitic infection in dogs is lacking. To evaluate the efficacy of oxfendazole, 11 out of the 21 client-owned dogs (21/123; 17.1%) positive for skin-dwelling O. lupi microfilariae (mfs), were enrolled in the efficacy study and were treated with oxfendazole (50 mg/kg) per OS once a day for 5 (G2) or 10 (G3) consecutive days or were left untreated (G1). The efficacy of oxfendazole in the reduction of O. lupi mfs was evaluated by microfilarial count and by assessing the percentage of mfs reduction and mean microfilaricidal efficacy, whereas the efficacy in the reduction of ocular lesions was evaluated by ultrasound imaging. All dogs where subjected to follow-ups at 30 (D30), 90 (D90) and 180 (D180) days post-treatment. The percentage of reduction of mfs was 78% for G2 and 12.5% for G3 at D180. The mean microfilaricidal efficacy of oxfendazole in the treatment of canine onchocercosis by O. lupi at D30, D90 and D180 was 41%, 81% and 90%, in G2 and 40%, 65% and 70%, in G3, respectively. Retrobulbar lesions did not reduce from D0 to D180 in control group (dogs in G1), whereas all treated dogs (in G2 and G3) had slightly decreased ocular lesions. Percentage of reduction of ocular lesions by ultrasound examination was 50% and 47.5% in G2 and G3 at D180, respectively. Despite the decrease in ocular lesions in all treated dogs (G2 and G3), oxfendazole was ineffective in reducing ocular lesions and skin-dwelling O. lupi mfs in treated dogs (G2 and G3) in a six-month follow-up period. Here we discuss the need for more reliable diagnostic techniques and efficient treatment protocols to better plan future intervention strategies.

Dataset Information

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

Publications

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

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