兔耳痒螨 var. 豚草亚种两丝氨酸蛋白酶抑制剂的分子特征及血清学诊断潜力。

Molecular characterization and serodiagnostic potential of two serpin proteins in Psoroptes ovis var. cuniculi.

机构信息

Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, Sichuan, People's Republic of China.

Mianyang Animal Disease Control Center, Mianyang, 621000, Sichuan, People's Republic of China.

出版信息

Parasit Vectors. 2020 Dec 11;13(1):620. doi: 10.1186/s13071-020-04501-8.

DOI:10.1186/s13071-020-04501-8

PMID:33308317

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7731772/

Abstract

BACKGROUND

Psoroptes ovis var. cuniculi is a common ectoparasite of wild and domestic rabbits worldwide that causes economically devastating losses in commercial rabbit husbandry and significantly affects the overall health of rabbits. Serine proteinase inhibitor (serpin) is present in almost all organisms that are involved in host-pathogen interactions, inflammatory responses, and reproductive development, among others. However, very little research has been carried out on P. ovis var. cuniculi serpins.

METHODS

Two serpin genes of P. ovis var. cuniculi (Pso c 27 and PsoSP2 cDNAs) were cloned and molecularly characterized. The transcriptional profiles and tissue localization of these two serpins in P. ovis var. cuniculi were investigated by quantitative real-time PCR and immunohistochemistry, respectively. The potential function of recombinant Pso c 27 and PsoSP2 (rPso c 27 and rPsoSP2) in the serodiagnosis of P. ovis var. cuniculi infestation in rabbits was evaluated using a newly devleoped indirect enzyme-linked immunosorbent assay.

RESULTS

Both the 523-residue Pso c 27 and the 240-residue PsoSP2 proteins contained typical serpin domains and signatures. Both Pso c 27and PsoSP2 cDNAs were expressed throughout the life-cycle; specifically, the cDNAs showed significantly higher expression in female mites than in larva, nymph, and male mites (Pso c 27: F = 1935.953, P < 0.0001; PsoSP2: F = 660.669, P < 0.0001). The native Pso c 27 and PsoSP2 proteins localized in the ovary and mouthparts of adult female mites, respectively. Compared to rPsoSP2, rPso c 27 showed better diagnostic efficiency, with higher values of sensitivity, specificity, and area under the receiver operating characteristic curve (rPso c 27 vs rPsoSP2: 96.0 vs 90.0%; 90.91 vs 78.18%; 0.988 vs 0.964, respectively). Moreover, rPso c 27 showed seropositivity in 80% of the rabbits as early as the 2 weeks post-infestation, prior to visible clinical signs and microscopy-positive of skin scrapings.

CONCLUSIONS

These results suggest that these two serpins may play essential roles in reproductive development, serum-feeding, and pathogenicity of P. ovis var. cuniculi. Compared to PsoSP2, Pso c 27 appears to be a potential antigen for serodiagnosis of P. ovis var. cuniculi infestation in rabbits, especially at the early stage of infestation.

摘要

背景

兔痒螨是一种常见的兔科动物外寄生虫，全球范围内的野生和家养兔都深受其害，给商业养兔业造成了巨大的经济损失，严重影响了兔群的整体健康。丝氨酸蛋白酶抑制剂（serpin）存在于几乎所有参与宿主-病原体相互作用、炎症反应和生殖发育等过程的生物中。然而，目前对兔痒螨的丝氨酸蛋白酶抑制剂研究甚少。

方法

本研究克隆并鉴定了兔痒螨 2 个丝氨酸蛋白酶抑制剂基因（Pso c 27 和 PsoSP2 cDNA），通过实时定量 PCR 和免疫组织化学技术分别研究了这两个丝氨酸蛋白酶抑制剂在兔痒螨中的转录谱和组织定位。通过新开发的间接酶联免疫吸附试验（ELISA）评估了重组 Pso c 27（rPso c 27）和 PsoSP2（rPsoSP2）在兔痒螨感染血清学诊断中的潜在功能。

结果

全长 523 个氨基酸的 Pso c 27 和全长 240 个氨基酸的 PsoSP2 蛋白均含有典型的丝氨酸蛋白酶抑制剂结构域和特征。这两个基因在整个生命周期中均有表达，在雌螨中表达水平显著高于幼虫、若螨和雄螨（Pso c 27：F=1935.953，P<0.0001；PsoSP2：F=660.669，P<0.0001）。天然 Pso c 27 和 PsoSP2 蛋白分别定位于成熟雌螨的卵巢和口器中。与 rPsoSP2 相比，rPso c 27 具有更好的诊断效率，其敏感性、特异性和受试者工作特征曲线下面积（rPso c 27 与 rPsoSP2：96.0%比 90.0%；90.91%比 78.18%；0.988 比 0.964）更高。此外，rPso c 27 在感染后 2 周即可在 80%的感染兔中检测到血清阳性，早于皮肤刮片可见临床症状和显微镜阳性。