Abstract
Bovine paratuberculosis (PTB) is caused by Mycobacterium avium subsp. paratuberculosis (MAP). The 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. The validation consisted of the assessment of 10 sera from PTB-infected and healthy bovines with different OD values. The 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). The ELISA using the polyprotein yielded an area under the ROC curve (AUC) of 0.9912 (95% CI, 0.9758–1.007; P < 0.0001). Moreover, for this 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%). These results demonstrate a very low cross-reactivity of the polyprotein with antibodies present in serum samples from animals infected with M. bovis. The designed polyprotein and the validated ELISA could be very useful for the specific identification of MAP-infected animals in herds.
Highlights
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 infection is by ingestion of contaminated milk, colostrum, or feces [3]
E protein sequences of the four antigens (MAP0038, MAP0209c, MAP2513c, and MAP1589c) of Mycobacterium avium subsp. paratuberculosis (MAP) K-10 selected for the design of the polyprotein were obtained from Uniprot database. e B-cell epitopes from the antigen sequences were predicted using the BepiPred-2.0 program [36]. e recombinant polyprotein, synthesized by Genscript (Jinsite Science and Technology, Nanjing, China), had 593 nucleotides and a fusion of four B epitopes with a six-His tag at the 3′ end of the sequence. e polyprotein was cloned in the vector pET-23a(+) and expressed in E. coli BL21 pLysS by induction of 1.0-L Luria Bertani (LB) broth cultures with 0.3 Mm isopropyl-β-d-thiogalactopyranoside (Sigma-Aldrich, Louis, MO) for 16 h at 20°C
According to previous results of our group and other publications, the antigens selected for the design of the polyprotein of this study were those only detected in sera from animals infected with MAP but not with M. bovis [39, 40]. e recombinant polyprotein was successfully expressed, as evidenced by a Western blot using an anti-His antibody (Figure 1(a))
Summary
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 infection is by ingestion of contaminated milk, colostrum, or feces [3]. 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. E entry of MAP is mediated by intestinal M cells and preferentially resides in phagosomes or early endosomes of host macrophages, predominately those associated with ileal Peyer’s patches [5]. Economic losses due to PTB are related to death, early elimination of animals, increased susceptibility to other infectious diseases, reduction of milk, meat, and reproductive yields, among others [10,11,12]. The association of MAP with Crohn’s disease (CD) in humans, which is an autoimmune disease related to chronic intestinal depletion, suggests a zoonotic relevance [13]
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