Antibody detection against Kunitz-type protein in Fasciola hepatica experimentally infected sheep using enzyme-linked immunosorbent assay (ELISA)

Abstract Fasciolosis is a parasitic disease considered as emerging and neglected by the WHO. Sheep are highly susceptible to this disease, and affected flocks experience decreased productivity due to increased mortality and reduced quality of their products such as wool and meat. To effectively control this disease, reliable and early diagnosis is essential for making decisions regarding the application of antiparasitics and/or the removal of affected animals. The current diagnosis of F. hepatica in sheep relies on the detection of parasite eggs in feces, a method that becomes reliable from week 10 post-infection. Consequently, there is a need for earlier diagnostic tools based on immune response. However, the obtaining of antigens for antibody detection has proven to be difficult and expensive. In this study, we compared the performance of recombinant F. hepatica Kunitz-type inhibitors (FhKT1.1, FhKT1.3, and FhKT4) in sera from experimentally F. hepatica-infected sheep six weeks post-infection with a synthetic Kunitz-type peptide (sFhKT) used as antigens by ELISA. Among these, FhKT1.1 showed the most promising diagnostic indicators, exhibiting high precision and low cross-reactivity, thus holding potential for standardized production. The results of our study demonstrated that the application of FhKT1.1 is a valuable tool for early-stage diagnosis of F. hepatica in sheep. Such an early diagnosis can aid in implementing timely interventions and effectively managing the disease in sheep populations

摘要：片形吸虫病（Fasciolosis）是一种被世界卫生组织（WHO）列为新发且被忽视的寄生虫病。绵羊对该疾病具有高度易感性，受感染羊群会因死亡率上升以及羊毛、肉类等产品品质下降而出现生产力降低的情况。若要有效防控该疾病，可靠的早期诊断是制定抗寄生虫药使用方案及（或）淘汰患病动物决策的关键前提。当前针对绵羊肝片形吸虫（F. hepatica）的诊断主要依赖粪便中寄生虫卵的检测，但该方法需在感染后第10周才能达到可靠的检测效果。因此，亟需开发基于免疫应答的早期诊断工具。然而，用于抗体检测的抗原获取曾被证明难度大且成本高昂。本研究将重组肝片形吸虫Kunitz型抑制剂（FhKT1.1、FhKT1.3及FhKT4）应用于感染后6周的实验感染绵羊血清，并与作为抗原的合成Kunitz型肽（sFhKT）开展酶联免疫吸附试验（ELISA）对比。结果显示，FhKT1.1展现出最优的诊断性能，兼具高精度与低交叉反应性，具备标准化生产的潜力。本研究结果表明，FhKT1.1可作为绵羊肝片形吸虫感染早期诊断的有效工具，此类早期诊断可助力及时采取干预措施，有效管控绵羊群体中的该疾病。