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

HERMIDA H; COLAVECCHIA S.; FERNÁNDEZ B.; MEREB G; MARTINEZ VIVOT M; SUHEVIC J; JAR AM; MUNDO SL.

Cancún

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

Latin American Association of Immunology

The diagnosis of paratuberculosis is made by identification of Mycobacterium avium subsp. paratuberculosis in the bacteriological culture of feces. However, this technique requires a minimum of 3 months. Therefore, indirect techniques (ELISA) are currently being 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 losses in animal production. In Argentina, the deer?s breeding fields has been increasing in the last years, and along with it, the demand for rapid and accurate diagnosis for this infection. The aim of this work was the production of a rabbit anti-deer polyclonal antibody and it application into an ELISA-PPA test. For this purpose, two rabbits were immunized subcutaneously with three doses of 1 mg/mL in PBS of deer gamma-globulin obtained by ammonium sulfate precipitation and emulsified with incomplete Freund?s adjuvant. Rabbit serum was precipitated, purified with protein A and characterized. Also titer and cross-reactions with other species were evaluated by ELISA. Two different ELISAs were designed using PPA as antigen. ELISA A was performed using the rabbit anti-deer produced and a HRP-conjugated goat anti-rabbit as secondary antibody. In ELISA B an AP-conjugated goat anti-deer IgG commercially available was used. In order to achieve the best conditions for both assays, different dilutions of known positive and negative deer sera, PPA and each antibody were tested. Samples of feces and sera (n=16), nine positive and seven negative, were evaluated; feces by culture and sera by ELISA A and B. Sensitivity, specificity and concordance strength between each ELISA and the fecal culture were analyzed by ROC curves and Kappa index using MedCalcStatistical Software. Subsequently, sera from a breeding field suspected of being infected with paratuberculosis (n=155) were evaluated by ELISA A and B. The obtained titer was >256000 and cross-reactions with other ruminant species were detected: 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 concordance strength 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 number of positive animals in the suspected herd (76.77% vs. 23.23%). The observed results demonstrated that the anti-deer polyclonal antibody produced could 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 species which would reduce the cost of the ELISA test.