Research Article Development and Validation of a Novel ELISA for the Specific DetectionofAntibodiesagainst Mycobacterium avium Subspecies paratuberculosis Based on a Chimeric Polyprotein RobertoDami´ an Moyano , 1 Magali Andrea Romero , 2 Mar´ ıa Alejandra Colombatti Olivieri , 1 Mar´ ıa Fiorella Alvarado Pinedo , 3 Gabriel Eduardo Traveria , 3 Mar´ ıa Isabel Romano , 1 and Mar´ ıa Natalia Alonso 1 1 Instituto de Biotecnolog´ ıa, Instituto Nacional de Tecnolog´ ıa Agropecuaria (INTA), Instituto de Agrobiotecnolog´ ıa y Biolog´ ıa Molecular (IABiMo), Consejo Nacional de Investigaciones Cient´ ıficas y T´ ecnicas (CONICET), Nicol´ as Repetto y Los Reseros, P.O. Box 1686, Hurlingham, Buenos Aires, Argentina 2 Consejo Nacional de Investigaciones Cient´ ıficas y T´ ecnicas (CONICET), Godoy Cruz 2290 (C1425FQB), CABA, Argentina 3 Centro de Diagn´ ostico e Investigaciones Veterinarias (CEDIVE) de la Facultad de Ciencias Veterinarias, Universidad de La Plata, Alvear 803, P.O. Box 7130, Chascomus, Buenos Aires, Argentina Correspondence should be addressed to Mar´ ıa Alejandra Colombatti Olivieri; colombatti.alejandra@inta.gob.ar Received 9 September 2021; Revised 27 November 2021; Accepted 8 December 2021; Published 29 December 2021 Academic Editor: Fulvio Riondato Copyright © 2021 Roberto Dami´ an Moyano et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Bovine paratuberculosis (PTB) is caused by Mycobacterium avium subsp. paratuberculosis (MAP). e optimization of detection tests specific for MAP is crucial to improve PTB control. In this work, we aimed to develop and validate a diagnostic tool based on an ELISA to specifically detect anti-MAP antibodies from bovine serum samples. For that purpose, we designed a recombinant polyprotein containing four specific antigens from MAP and optimized the ELISA. e validation consisted of the assessment of 10 sera from PTB-infected and healthy bovines with different OD values. e diagnostic performance of the polyprotein-ELISA was evaluated by testing 130 bovine serum samples (47 healthy, 48 MAP-infected, and 35 M. bovis-infected bovines). e ELISA usingthepolyproteinyieldedanareaundertheROCcurve(AUC)of0.9912(95%CI,0.9758–1.007; P < 0.0001).Moreover,forthis ELISA, the cut-off selected from the ROC curve based on the point with a sensitivity of 95.56% (95% CI, 0.8485–0.9946) and specificity of 97.92 (95% CI, 0.8893–0.9995) was 0.3328. Similar results were obtained with an ELISA using the commercial Paratuberculosis Protoplasmatic Antigen (PPA). However, the ELISA with the polyprotein antigen showed a better performance against sera from animals infected with Mycobacterium bovis compared to the ELISA with PPA: lower cross-reactivity (2.85% versus 25.71%). ese results demonstrate a very low cross-reactivity of the polyprotein with antibodies present in serum samples from animals infected with M. bovis. e designed polyprotein and the validated ELISA could be very useful for the specific identification of MAP-infected animals in herds. 1.Introduction Bovine paratuberculosis (PTB) or Johne’s disease, an endemic disease in many parts of the world, is a highly contagious chronic progressive granulomatous enteritis responsible for considerable losses to livestock and associated industries [1], whose etiologic agent is Mycobacterium avium subsp. paratuberculosis (MAP) [2]. e most common route of in- fection is by ingestion of contaminated milk, colostrum, or feces [3]. Calves up to 6 months of age are at higher risk of getting infected but the risk drops afterwards [4]. e entry of MAP is mediated by intestinal M cells and preferentially re- sides in phagosomes or early endosomes of host macrophages, predominately those associated with ileal Peyer’s patches [5]. Hindawi Veterinary Medicine International Volume 2021, Article ID 7336848, 10 pages https://doi.org/10.1155/2021/7336848