Diagnostic value of recombinant nanoluciferase fused Toxoplasma gondii antigens in Luciferase-linked Antibody Capture Assay (LACA) for Toxoplasma infection in pigs.

Toxoplasmosis is a widespread protozoan zoonosis. Since ingesting undercooked meat harboring Toxoplasma gondii cyst is considered one of the major transmission routes to humans, the screening of T. gondii in meat-producing animals can reduce the risk of food-borne toxoplasmosis in humans. Among serological diagnostic methods, Luciferase-linked Antibody Capture Assay (LACA) has been found to be a promising platform with high sensitivity and specificity. In this study, we aimed to evaluate recombinant nanoluciferase fused-T. gondii antigens (rNluc-GRA6, rNluc-GRA7, rNluc-GRA8 and rNluc-BAG1) for their potential use in LACA for pigs. As a result, the sensitivity of GRA6-, GRA7-, GRA8- and BAG1-LACA were 70.0%, 80.0%, 80.0% and 30.0% with specificity 87.0%, 81.5%, 74.1% and 50.0%, respectively. The cocktail LACA using a mixture of rNluc-GRA6, rNluc-GRA7 and rNluc-GRA8 indicated higher sensitivity (90.0%) and a similar specificity (96.3%) in comparison with the commercial ELISA kit. Compared to the Dye-Test as a reference test, cocktail LACA showed strong agreement (kappa value=0.811) when we assessed pig sera collected at the slaughterhouse. In addition, we also successfully established the rapid LACA format for the detection of Toxoplasma infection in pigs (called Rapid-LACA) in which the test could be performed within 30 min. In Rapid-LACA, the protein A pre-coated/blocked plates could be preserved at -30°C, 4°C or room temperature conditions for at least two months without compromising on the quality of assay.

A novel luciferase-linked antibody capture assay (LACA) for the diagnosis of Toxoplasma gondii infection in chickens.

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes the most common parasitic zoonosis worldwide in multiples species of mammals and birds. Although free-range chickens may play a role as an important reservoir for T. gondii, there is no reliable and commercially available diagnostic test for this disease in chickens. In this study, we aimed to develop a novel Luciferase-linked Antibody Capture Assay (LACA) for the serodiagnosis of Toxoplasma infection in chickens. Recombinant nanoluciferase fused-T. gondii dense granule antigen 8 (rNluc-GRA8) was produced and applied to LACA assay as a diagnostic antigen. GRA8-LACA was tested with the sera from uninfected and experimentally infected chickens with T. gondii and other parasitic pathogens and showed unexpectedly high sensitivity (90.5%) and specificity (95.4%). Interestingly, E. coli lysate expressing rNluc-GRA8 could be applied in GRA8-LACA with 85.7% sensitivity and an increased specificity (96.9%) that gave better diagnostic performance compared to conventional ELISA. We applied our diagnostic system to examine 267 free-range chicken sera collected from 12 farms and 100 closed-house broiler chicken sera from local poultry abattoirs. The overall seroprevalence of toxoplasmosis in free-range chickens was 10.9% (95% CI: 10.6%-11.1%), while no positive case was found in broiler chickens. GRA8-LACA could be a useful diagnostic technique for T. gondii infection in chickens. The detection of T. gondii seropositive chickens in this study warns a potential risk of Toxoplasma transmission by the consumption of raw or undercooked chicken meat.