Production and development of an ELISA-PPA for the diagnosis of Mycobacterium avium subsp. paratuberculosis in red deer (Cervus elaphus)

HERMIDA H; COLAVECCHIA, SB.; FERNÁNDEZ B.; MEREB G; MARTINEZ VIVOT; SUHEVIC J; JAR, A. M.; MUNDO SL.

Cancún

Congreso; XII Congress of the Latin American Association of Immunology y XXIII Congress of the Mexican Society of Immunology.; 2018

Latin American Association of Immunology

The diagnosis of paratuberculosis is made by identification of Mycobacterium aviumsubsp. paratuberculosis in the bacteriological culture of feces. However, this techniquerequires a minimum of 3 months. Therefore, indirect techniques (ELISA) are currentlybeing used to reduce the diagnosis time. Protoplasmic paratuberculosis antigen (PPA)is the antigen recommended by the OIE (World Organisation for Animal Health).Paratuberculosis affects domestic and wild ruminants, generating great economic lossesin animal production. In Argentina, the deer?s breeding fields has been increasing inthe last years, and along with it, the demand for rapid and accurate diagnosis for thisinfection.The aim of this work was the production of a rabbit anti-deer polyclonal antibody andit application into an ELISA-PPA test. For this purpose, two rabbits were immunizedsubcutaneously with three doses of 1 mg/mL in PBS of deer gamma-globulin obtainedby ammonium sulfate precipitation and emulsified with incomplete Freund?s adjuvant.Rabbit serum was precipitated, purified with protein A and characterized. Also titerand cross-reactions with other species were evaluated by ELISA. Two different ELISAswere designed using PPA as antigen. ELISA A was performed using the rabbit anti-deerproduced and a HRP-conjugated goat anti-rabbit as secondary antibody. In ELISA B anAP-conjugated goat anti-deer IgG commercially available was used. In order to achievethe best conditions for both assays, different dilutions of known positive and negativedeer sera, PPA and each antibody were tested. Samples of feces and sera (n=16), ninepositive and seven negative, were evaluated; feces by culture and sera by ELISA Aand B. Sensitivity, specificity and concordance strength between each ELISA and thefecal culture were analyzed by ROC curves and Kappa index using MedCalcStatistical Software. Subsequently, sera from a breeding field suspected of being infected withparatuberculosis (n=155) were evaluated by ELISA A and B.The obtained titer was >256000 and cross-reactions with other ruminant species weredetected: cattle, sheep, llama and goat (>64000). ELISA A obtained a greater sensitivity(85.71%) and specificity (88.89%) than ELISA B (77.78% and 57.00%). The concordancestrength with fecal culture was ?good? k=0.62 ± 0.19 in ELISA A. On the other hand,ELISA B threw a ?weak? value k=0.35 ± 0.24. ELISA A also identified a larger numberof positive animals in the suspected herd (76.77% vs. 23.23%).The observed results demonstrated that the anti-deer polyclonal antibody producedcould be useful in serological diagnostic tests in deer with high sensitivity and specificity.In addition, this antibody would allow us to utilize the same reagent in different specieswhich would reduce the cost of the ELISA test.