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

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 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.

Early detection of Mycobacterium avium subsp. paratuberculosis infection in cattle with multiplex-bead based immunoassays

“…Since the first complete MAP genome sequence was published [ 9 ], many studies with recombinant MAP proteins have been conducted to identify potential candidates for use as diagnostic antigens that could distinguish animals with mild or early MAP infection from those uninfected [ 10 – 16 ]. We recently screened a set of well-characterized serum samples using a whole proteome microarray from Mycobacterium tuberculosis (MTB), and several promising candidate antigens were identified from these studies as immunogenic during MAP infection [ 17 ]. These antigens need to be further evaluated for the development of a high-throughput, diagnostic immunoassay.…”

“…Here, we describe the development of a multiplex immunoassay for simultaneous detection of antibodies specific to six candidate recombinant MAP proteins. Five of these proteins (MAP1272c, MAP1569, MAP2609, MAP2942c and MAP1201c+2942c fusion protein) were selected because they displayed the highest levels of sensitivity and specificity in our previous protein array studies [ 17 ]. Additionally, MAP2121c was selected based on previous studies that showed significant reactivity to samples from infected animals in previous ELISA studies [ 10 , 23 ] although it was not shown in the MTB array due to the absence of an ortholog in MTB [ 17 ].…”

Characterization of Ethanol Extracted Cell Wall Components of Mycobacterium avium Subsp. paratuberculosis

“…The two Map recombinant proteins identified in the EtOH extracts were tested previously for the ability to discriminate JD cows from healthy animals using protein arrays [27]. MAP_2698c was not immunogenic in those studies and MAP_0585 showed some promise as an antigen since it ranked in the upper half of all proteins examined.…”

Interplay of primary bovine lymphocytes and Mycobacterium avium subsp. paratuberculosis shows distinctly different proteome changes and immune pathways in host-pathogen interaction

“…Paratuberculosis, also referred to as Johne’s disease, is world-wide endemic; no country or region has been found to be free of this disease (Li et al, 2016). Affected ruminants go through a long asymptomatic subclinical phase in which infection cannot reliably be detected by standard diagnostic tests (Hobmaier et al, 2019; Li et al, 2017). These subclinically infected animals can already shed MAP, thereby contaminating dairy products or meat (Sweeney, 2011).…”