Dataset Information

Identification of Sero-Diagnostic Antigens for the Early Diagnosis of Johne's Disease using MAP Protein Microarrays.

ABSTRACT: Considerable effort has been directed toward controlling Johne's disease (JD), a chronic granulomatous intestinal inflammatory disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) in cattle and other ruminants. However, progress in controlling the spread of MAP infection has been impeded by the lack of reliable diagnostic tests that can identify animals early in the infection process and help break the transmission chain. To identify reliable antigens for early diagnosis of MAP infection, we constructed a MAP protein array with 868 purified recombinant MAP proteins, and screened a total of 180 well-characterized serum samples from cows assigned to 4 groups based on previous serological and fecal test results: negative low exposure (NL, n?=?30); negative high exposure (NH, n?=?30); fecal-positive, ELISA-negative (F?+?E-, n?=?60); and both fecal- and ELISA-positive (F?+?E+, n?=?60). The analyses identified a total of 49 candidate antigens in the NH, F?+?E-, and F?+?E+ with reactivity compared with the NL group (p?

SUBMITTER: Li L

PROVIDER: S-EPMC6879513 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Identification of Sero-Diagnostic Antigens for the Early Diagnosis of Johne's Disease using MAP Protein Microarrays.

Li Lingling L Bannantine John P JP Campo Joseph J JJ Randall Arlo A Grohn Yrjo T YT Schilling Megan A MA Katani Robab R Radzio-Basu Jessica J Easterling Laurel L Kapur Vivek V

Scientific reports 20191126 1

Considerable effort has been directed toward controlling Johne's disease (JD), a chronic granulomatous intestinal inflammatory disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) in cattle and other ruminants. However, progress in controlling the spread of MAP infection has been impeded by the lack of reliable diagnostic tests that can identify animals early in the infection process and help break the transmission chain. To identify reliable antigens for early diagnosis of MAP in ...[more]

PMID: 31772281

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Project description: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 and causes serious animal health problems and significant economic loss in domesticated ruminants throughout the world. Since serological detection of MAP infected animals during the early stages of infection remains challenging due to the low sensitivity of extant assays, we screened 180 well-characterized serum samples using a whole proteome microarray from Mycobacterium tuberculosis (MTB), a close relative of MAP. Based on extensive testing of serum and milk samples, fecal culture and qPCR for direct detection of MAP, the samples were previously 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+). Of the 740 reactive proteins, several antigens were serologically recognized early but not late in infection, suggesting a complex and dynamic evolution of the MAP humoral immune response during disease progression. Ordinal logistic regression models identified a subset of 47 candidate proteins with significantly different normalized intensity values (p<0.05), including 12 in the NH and 23 in F+E- groups, suggesting potential utility for the early detection of MAP infected animals. Next, the diagnostic utility of four MAP orthologs (MAP1569, MAP2942c, MAP2609, and MAP1272c) was assessed and reveal moderate to high diagnostic sensitivities (range 48.3% to 76.7%) and specificity (range 96.7% to 100%), with a combined 88.3% sensitivity and 96.7% specificity. Taken together, the results of our analyses have identified several candidate MAP proteins of potential utility for the early detection of MAP infection, as well individual MAP proteins that may serve as the foundation for the next generation of well-defined serological diagnosis of JD in cattle.

Project description:Johne's disease, a chronic gastrointestinal inflammatory disease caused by Mycobacterium avium subspecies paratuberculosis, is endemic in dairy cattle and other ruminants worldwide and remains a challenge to diagnose using traditional serological methods. Given the close phylogenetic relationship between M. avium subsp. paratuberculosis and the human pathogen Mycobacterium tuberculosis, here, we applied a whole-proteome M. tuberculosis protein array to identify seroreactive and diagnostic M. avium subsp. paratuberculosis antigens. A genome-scale pairwise analysis of amino acid identity levels between orthologous proteins in M. avium subsp. paratuberculosis and M. tuberculosis showed an average of 62% identity, with more than half the orthologous proteins sharing >75% identity. Analysis of the M. tuberculosis protein array probed with sera from M. avium subsp. paratuberculosis-infected cattle showed antibody binding to 729 M. tuberculosis proteins, with 58% of them having ?70% identity to M. avium subsp. paratuberculosis orthologs. The results showed that only 4 of the top 40 seroreactive M. tuberculosis antigens were orthologs of previously reported M. avium subsp. paratuberculosis antigens, revealing the existence of a large number of previously unrecognized candidate diagnostic antigens. Enzyme-linked immunosorbent assay (ELISA) testing of 20 M. avium subsp. paratuberculosis recombinant proteins, representing reactive and nonreactive M. tuberculosis orthologs, further confirmed that the M. tuberculosis array has utility as a screening tool for identifying candidate antigens for Johne's disease diagnostics. Additional ELISA testing of field serum samples collected from dairy herds around the United States revealed that MAP2942c had the strongest seroreactivity with Johne's disease-positive samples. Collectively, our studies have considerably expanded the number of candidate M. avium subsp. paratuberculosis proteins with potential utility in the next generation of rationally designed Johne's disease diagnostic assays.

Project description:The function and extracellular location of cell envelope proteins make them attractive candidates for developing vaccines against bacterial diseases, including challenging drug-resistant pathogens, such as Neisseria gonorrhoeae A proteomics-driven reverse vaccinology approach has delivered multiple gonorrhea vaccine candidates; however, the biological functions of many of them remain to be elucidated. Herein, the functions of six gonorrhea vaccine candidates-NGO2121, NGO1985, NGO2054, NGO2111, NGO1205, and NGO1344-in cell envelope homeostasis were probed using phenotype microarrays under 1,056 conditions and a ?bamE mutant (?ngo1780) as a reference of perturbed outer membrane integrity. Optimal growth conditions for an N. gonorrhoeae phenotype microarray assay in defined liquid medium were developed, which can be useful in other applications, including rapid and thorough antimicrobial susceptibility assessment. Our studies revealed 91 conditions having uniquely positive or negative effects on one of the examined mutants. A cluster analysis of 37 and 57 commonly beneficial and detrimental compounds, respectively, revealed three separate phenotype groups: NGO2121 and NGO1985; NGO1344 and BamE; and the trio of NGO1205, NGO2111, and NGO2054, with the last protein forming an independent branch of this cluster. Similar phenotypes were associated with loss of these vaccine candidates in the highly antibiotic-resistant WHO X strain. Based on their extensive sensitivity phenomes, NGO1985 and NGO2121 appear to be the most promising vaccine candidates. This study establishes the principle that phenotype microarrays can be successfully applied to a fastidious bacterial organism, such as N. gonorrhoeae IMPORTANCE Innovative approaches are required to develop vaccines against prevalent and neglected sexually transmitted infections, such as gonorrhea. Herein, we have utilized phenotype microarrays in the first such investigation into Neisseria gonorrhoeae to probe the function of proteome-derived vaccine candidates in cell envelope homeostasis. Information gained from this screening can feed the vaccine candidate decision tree by providing insights into the roles these proteins play in membrane permeability, integrity, and overall N. gonorrhoeae physiology. The optimized screening protocol can be applied in investigations into the function of other hypothetical proteins of N. gonorrhoeae discovered in the expanding number of whole-genome sequences, in addition to revealing phenotypic differences between clinical and laboratory strains.

Project description:BackgroundThe simplicity and potential of minimal invasive testing using serum from patients make auto-antibody based biomarkers a very promising tool for use in diagnostics of cancer and auto-immune disease. Although several methods exist for elucidating candidate-protein markers, immobilizing these onto membranes and generating so called macroarrays is of limited use for marker validation. Especially when several hundred samples have to be analysed, microarrays could serve as a good alternative since processing macro membranes is cumbersome and reproducibility of results is moderate.MethodsCandidate markers identified by SEREX (serological identification of antigens by recombinant expression cloning) screenings of brain and lung tumour were used for macroarray and microarray production. For microarray production recombinant proteins were expressed in E. coli by autoinduction and purified His-tag (histidine-tagged) proteins were then used for the production of protein microarrays. Protein arrays were hybridized with the serum samples from brain and lung tumour patients.ResultMethods for the generation of microarrays were successfully established when using antigens derived from membrane-based selection. Signal patterns obtained by microarrays analysis of brain and lung tumour patients' sera were highly reproducible (R = 0.92-0.96). This provides the technical foundation for diagnostic applications on the basis of auto-antibody patterns. In this limited test set, the assay provided high reproducibility and a broad dynamic range to classify all brain and lung samples correctly.ConclusionProtein microarray is an efficient means for auto-antibody-based detection when using SEREX-derived clones expressing antigenic proteins. Protein microarrays are preferred to macroarrays due to the easier handling and the high reproducibility of auto-antibody testing. Especially when using only a few microliters of patient samples protein microarrays are ideally suited for validation of auto-antibody signatures for diagnostic purposes.

Dataset Information

Identification of Sero-Diagnostic Antigens for the Early Diagnosis of Johne's Disease using MAP Protein Microarrays.

Publications

Identification of Sero-Diagnostic Antigens for the Early Diagnosis of Johne's Disease using MAP Protein Microarrays.

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