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

AHUMADA, MARÍA; GODINO, AGUSTINA; GUASCONI, LORENA; DEHEZA, CARLA; AMARANTO, MARILLA; PRUZZO, CESAR IVÁN; VITULLI-MOYA, GABRIEL; CHIAPELLO, LAURA; CARRIZO, MARÍA E.; BARRA, JOSÉ LUIS; CERVI, LAURA

San Luis

Congreso; LXXI Reunión Anual de la Sociedad Argentina de Inmunología (SAI); 2023

Sociedad Argentina de Inmunología (SAI)

Fasciolosis is a parasitic disease considered as emerging and neglected by the WHO. Sheep are highly susceptible to this disease showing a 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 (Fh) in sheep relies on the detection of parasite eggs in feces, a method that becomes reliable from week 10 postinfection. 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. Kunitz-type (KT) protease inhibitors are low molecular weight proteins with serine protease inhibitor activity. They have recently been described to be involved in the regulation of major parasite and/or host-secreted cathepsin L-like cysteine proteases. The aim of this work was to compare the performance of recombinant proteins (rFhKT) belonging to the family of FhKT inhibitors (FhKT1.1, FhKT1.3 and FhKT4) produced as fusion proteins with a synthetic (sFhKT) in a ELISA test for the diagnosis of F. hepatica in sheep. We evaluated the antibodies levels using a number of samples collected from experimentally F. hepatica-infected sheep six weeks after infection. Among the proteins used, FhKT1.1 showed the most promising diagnostic antigen, exhibiting high precision and low cross-reactivity, thus holding potential for standardized production.The precision of the diagnostic assay using FhKT was evaluated by determining the sensitivity, specificity, predictive values, likelihood ratio (LR) and kappa index. The specificity and sensitivity parameters calculated by the Wilson-Brown method showed thatthe best combination for both parameters is obtained using FhKT1.1(Sensibility (Se)0.83 and Specificity (Sp) 0.92, followed by sFhKT, FhKT4 and FhKT1.3.The kappa index was used to estimate the concordance between the results obtained by coproparasitological diagnosis (gold standard) and ELISA using sFhKT and rFhKTs. The highest concordance (Kappa index value= 0.91) was obtained for FhKT1.1, followed by sFhKT, FhKT4, and FhKT1.3. 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.