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

Onchocerca lupi 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 with O. lupi reactive sera and mass spectrometry. Onchocerca lupi major antigen protein (Ol-MJA) and paramyosin (Ol-PARA) were identified as potential biomarkers for serodiagnosis. Linear epitopes were scanned using high-density peptide microarray. Sera collected from dogs infected with O. lupi and healthy 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 (Achontochelonema reconditum and Cercopithifilaria bainae). Six antigenic peptides, three for Ol-MJA and for Ol-PARA, respectively, were selected as potential candidate biomarkers 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.

卢氏盘尾丝虫（Onchocerca lupi）是一种寄生于家养食肉动物与人类的丝虫。成年线虫通常定位于感染宿主的巩膜下层或眼球后眶间隙，而微丝蚴则分布于皮肤组织内。因此，卢氏盘尾丝虫的诊断需通过对皮肤活检样本中的微丝蚴进行显微镜检查及/或分子检测，或通过手术摘除感染宿主眼部组织中的成虫来完成。目前亟需适用于犬卢氏盘尾丝虫感染诊断的非侵入性检测工具。本研究结合免疫印迹（immunoblotting）与质谱（mass spectrometry）技术开展了免疫蛋白质组学分析，鉴定出卢氏盘尾丝虫主要抗原蛋白（Ol-MJA）与副肌球蛋白（Ol-PARA），可作为血清学诊断的潜在生物标志物。研究借助高密度肽微阵列（high-density peptide microarray）扫描线性表位，通过使用卢氏盘尾丝虫感染犬血清与健康对照血清，共鉴定出11条免疫优势抗原肽（其中Ol-MJA相关7条，Ol-PARA相关4条）。随后，研究利用仅感染犬类主要丝虫的犬血清对上述肽段进行验证，所涉丝虫包括人畜共患型的匐行恶丝虫（Dirofilaria repens）、犬心丝虫（Dirofilaria immitis），以及非人畜共患型的隐管吸吮线虫（Achontochelonema reconditum）与贝恩氏猴丝虫（Cercopithifilaria bainae）。最终筛选出6条抗原肽（Ol-MJA与Ol-PARA各3条），作为犬卢氏盘尾丝虫感染血清学检测的潜在候选生物标志物。本研究报道的分子与蛋白质组学数据集，可为卢氏盘尾丝虫相关研究提供宝贵资源，助力开发针对犬盘尾丝虫病的新型干预手段，包括药物、疫苗与诊断试剂。