Huertas Lopez, AnaSánchez Sánchez, RobertoDiezma Díaz, CarlosÁlvarez García, GemaMartínez Carrasco, CarlosMartínez Subiela, SilviaCerón, José Joaquín2024-12-032024-12-032022Huertas-López, A., Sánchez-Sánchez, R., Diezma-Díaz, C., Álvarez-García, G., Martínez-Carrasco, C., Martínez-Subiela, S., & Cerón, J. J. (2022). Detection of anti-Neospora caninum antibodies in sheep's full-cream milk by a time-resolved fluorescence immunoassay. Veterinary parasitology, 301, 109641. https://doi.org/10.1016/j.vetpar.2021.1096410304-401710.1016/j.vetpar.2021.109641https://hdl.handle.net/20.500.14352/111995JUSTIFICACIÓN DE AUTORES: Huertas-López, Ana: Conceptualization; Formal analysis; Investigation; Methodology; Validation; Roles/Writing - original draft; Writing - review & editing. Sánchez-Sánchez, Roberto: Data curation; Investigation; Methodology; Writing - review & editing. Diezma-Díaz, Carlos: Data curation; Investigation; Methodology; Writing - review & editing. Álvarez-García, Gema: Conceptualization; Funding acquisition; Project administration; Supervision; Validation; Visualization; Writing - review & editing. Martínez-Carrasco, Carlos: Conceptualization; Project administration; Supervision; Validation; Visualization; Writing - review & editing. Martínez-Subiela, Silvia: Conceptualization; Formal analysis; Investigation; Methodology; Software; Supervision; Validation; Visualization; Writing - review & editing. Cerón, José Joaquín: Conceptualization; Funding acquisition; Project administration; Supervision; Validation; Visualization; Writing - review & editing.Ovine neosporosis, caused by the Apicomplexan parasite Neospora caninum, leads to reproductive failure worldwide. Nowadays, there is a trend to develop diagnostic techniques using non-invasive samples, such as milk, in order to reduce animal stress, sample collection effort, and costs. The objective of this study was to develop and validate a highly sensitive and specific serological technique, based on a time resolved-fluorescence immunoassay using a N. caninum GRA7 antigen (GRA7-TRFIA), for the detection of anti-N. caninum immunoglobulins G on sheep’ full-cream milk samples. An analytical validation was performed, including intra- and inter-assay precision, analytical sensitivity and accuracy. The diagnostic performance of the assay was evaluated by studying the positive-negative discrimination by Mann Whitney U tests. In additon optimal cut-offs, diagnostic sensitivity and specificity, and areas under the curve were calculated by three Receiver Operating Curve (ROC) analyses, using GRA7-TRFIA and a N. caninum tachyzoite soluble extract-based ELISA (NcSALUVET-ELISA) in blood sera, and the coinciding results of both techniques, as reference techniques. Moreover, Spearman’s correlation of GRA7-TRFIA in milk with the techniques in sera and agreement (kappa values) were also estimated. GRA7-TRFIA for milk samples showed an adequate precision, with high analytical sensitivity and accuracy. Regarding ROC analyses, at the optimal cut-offs, the diagnostic sensitivity and specificity were more than 90 % in all cases. In addition, GRA7-TRFIA values in milk were more positively correlated to GRA7-TRFIA values in blood sera than in the case of values obtained with NcSALUVET-ELISA. GRA7-TRFIA in milk showed an almost perfect agreement with GRA7-TRFIA in blood sera (kappa = 0.98) and with the coinciding results of GRA7-TRFIA and NcSALUVET in blood sera (kappa = 1.00), while it has a substantial agreement with NcSALUVET-ELISA (kappa = 0.69). In the light of these results, GRA7-TRFIA in full-cream milk samples is a highly sensitive technique that could be used for screening anti-N. caninum antibodies in sheep flocks.engAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/journal article1873-2550https://doi.org/10.1016/j.vetpar.2021.10964134979475https://www.sciencedirect.com/science/article/pii/S0304401721003010open access636.09Neospora caninumFull-cream milkSerologySheepTime-resolved fluorescence immunoassayCiencias Biomédicas24 Ciencias de la Vida