Identification of sero-reactive antigens for the early diagnosis of Johne's disease in cattle

Authors: LI, LINGLING - Pennsylvania State University; Bannantine, John; CAMPO, JOSEPH - Antigen Discovery, Inc; RANDALL, ARLO - Antigen Discovery, Inc; GROHN, YRJO - Pennsylvania State University; KATANI, ROBAB - Pennsylvania State University; SCHILLING, MEGAN - Pennsylvania State University; RADZIO-BASU, JESSICA - Pennsylvania State University; KAPUR, VIVEK - Pennsylvania State University

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/22/2017
Publication Date: 9/1/2017
Citation: Li, L., Bannantine, J.P., Campo, J.J., Randall, A., Grohn, Y.T., Katani, R., Schilling, M., Radzio-Basu, J., Kapur, V. 2017. Identification of sero-reactive antigens for the early diagnosis of Johne's disease in cattle. PLoS One. 12(9). https://doi.org/10.1371/journal.pone.0184373.
DOI: https://doi.org/10.1371/journal.pone.0184373

Interpretive Summary: This study builds on a previous study just published by our group where we used the Mycobacterium tuberculosis protein array to screen for antigens useful in Johne's disease diagnostics. That study identified useful antigens and now this study examined them more closely with an expanded set of serum and milk samples from Johne's disease cows. The overall goal is to use some combination of antigens in a multiplex-type assay to accurately identify infected cows. Because the sample sets we chose were so well characterized, we could separate them into early and late stage disease samples. With this approach, we identified proteins strongly reacting at early disease stages. We concluded the study by validating the ELISA test using an expanded serum set and a four-antigen combination. We found this combination worked best and can be used to build a multiplex test. This study is of primary interest to other researchers working in the field, veterinarians, and stakeholders.

Technical Abstract: Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease (JD), a chronic intestinal inflammatory disease of cattle and other ruminants. JD has a high herd prevalence rate and is recognized as a serious animal health problem and a cause of significant economic loss in domesticated ruminants throughout the world. Detection of MAP infected animals within herds, particularly during the early stages of infection, remains particularly challenging due to low sensitivity associated with extant assays. To address this challenge, we screened 180 well-characterized serum samples that were from the Johne’s Disease Integrated Program (JDIP) Diagnostic Standards Sample Collection using a whole proteome microarray from Mycobacterium tuberculosis (MTB), a closely related pathogen to MAP, containing ~4,000 proteins covering 97% of the entire genome. Prior to screening, the animals (originating from 16 dairy herds across 4 states) were evaluated using commercially available ELISAs for serum and milk samples, fecal culture and qPCR for direct detection of MAP and assigned to one of 4 groups: negative low exposure (n=30, NL); negative high exposure (n=30, NH); fecal positive, ELISA negative (n=60, F+E-); and fecal positive, ELISA positive (n=60, F+E+). Significantly reactive proteins were determined in groups NH, F+E-, and F+E+ based on comparison of their normalized reactivity intensities to that in the NL group (P<0.05). A total of 73 sero-reactive MAP orthologs were identified across three designated groups including 19 in the NH group, 30 in F+E-, and 53 in F+E+, which were unique in a group or shared between the groups. The diagnostic utility of the majority of orthologs identified, particularly in the NH and F+E- groups, have not been previously described. These antigenic proteins may be of considerable utility for the detection of MAP infection during early and middle stages of infection, which are currently very difficult to diagnose using traditional serological or microbiological approaches. ELISA testing on selected MAP recombinant proteins representing significantly reactive MTB orthologs displayed strong correlation to serological reactivity in the MTB array. Overall, the results of our study suggest that the MTB protein microarrays provide a powerful tool for the identification of potentially diagnostic MAP antigens, particularly during early stages of disease.