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 < 0.01), a majority of which have not been previously identified. While some of the antigens were identified as reactive in only one of the groups, others showed reactivity in multiple groups, including NH (n = 28), F + E− (n = 26), and F + E+ (n = 17) groups. Using combinations of top reactive antigens in each group, the results reveal sensitivities of 60.0%, 73.3%, and 81.7% in the NH, F + E−, and F + E+, respectively at 90% specificity, suggesting that early detection of infection in animals may be possible and enable better opportunities to reduce within herd transmission that may be otherwise missed by traditional serological assays that are biased towards more heavily infected animals. Together, the results suggest that several of the novel candidate antigens identified in this study, particularly those that were reactive in the NH and F + E− groups, have potential utility for the early sero-diagnosis of MAP infection.

Whole-Genome Analysis of Mycobacterium avium subsp. paratuberculosis IS900Insertions Reveals Strain Type-Specific Modalities

“…Strain K-10 ( Li et al, 2005 , 2019 ), Telford ( Brauning et al, 2019 ) and S397 ( Bannantine et al, 2012 ) were included in this study as references of the major lineages that have emerged during the evolution of Map . Isolates were propagated on slopes of modified Middlebrook 7H11 supplemented with 20% (vol/vol) heat inactivated newborn calf serum, 2.5% (vol/vol) glycerol, 2 mM asparagine, 10% (vol/vol) Middlebrook oleic acid-albumin- dextrose-catalase (OADC) enrichment medium (Becton Dickinson, Oxford, Oxfordshire, United Kingdom), Selectatabs (code MS 24; MAST Laboratories Ltd., Merseyside, United Kingdom), and 2 μg ml-1 mycobactin J (Allied Monitor, Fayette, MO, United States).…”

Draft Genome Sequences of 142 Mycobacterium avium subsp. paratuberculosis Strains Isolated from Naturally Infected Dairy Cattle

“…The metrics of the assembled contigs were collected using QUAST v5.0.2 ( 8 ), and the contigs were annotated using Prokka v1.14.6 ( 9 ) with M. avium subsp. paratuberculosis K-10 as the reference annotation strain ( 10 , 11 ). Prodigal ( 12 ) was pretrained using the complete genome sequence of strain K-10.…”

Biomarkers as diagnostic tools for mycobacterial infections in cattle

“…In recent years, studies on host-or pathogen-related biomarkerbased diagnostic methods have been conducted to overcome such problems as low sensitivity and specificity. Various diagnostic pathogen-derived biomarkers have been identified for the development of diagnostic methods for the detection of early stages of infection (Eda et al, 2006;Shin et al, 2008;Nagata et al, 2013;Li et al, 2017Li et al, , 2019. Eda and colleagues developed ELISA based on the surface antigen of MAP for the diagnosis of paratuberculosis.…”

“…Additionally, three antigens (MAP1569, MAP2942c, and MAP2609) significantly discriminate fecal PCR-positive and ELISA-negative animals from uninfected animals, indicating high diagnostic value for detecting the early stage of MAP infection (Li et al, 2017). Furthermore, these researchers constructed a MAP protein array with 868 purified recombinant MAP proteins and tested 180 serum samples from cows classified into four groups based on previous serum ELISA and fecal PCR tests (Li et al, 2019). A total of 49 candidate antigens were identified in the different stages of infection groups compared with the uninfected group.…”

“…A total of 49 candidate antigens were identified in the different stages of infection groups compared with the uninfected group. These researchers evaluated the diagnostic potential of MAP antigens with combinations of the top reactive antigens in each group, and the sensitivities were 60.0, 73.3, and 81.7% in the negative high-exposure group, fecal-positive and ELISAnegative group, and fecal-positive and ELISA-positive group, respectively, with 90% specificity indicating the possibility of detection for the early stage of MAP infection (Li et al, 2019).…”