Novel Secreted Antigens of Mycobacterium paratuberculosis as Serodiagnostic Biomarkers for Johne's Disease in Cattle

Abstract

Johne's disease is a chronic gastroenteritis of cattle caused by Mycobacterium avium subsp. paratuberculosis that afflicts 40% of dairy herds worldwide. M. avium subsp. paratuberculosis-infected cattle can remain asymptomatic for years while transmitting the pathogen via fecal contamination and milk. Current serodiagnosis with enzyme-linked immunosorbent assays (ELISAs) fails to detect asymptomatic M. avium subsp. paratuberculosis-infected cattle due to the use of poorly defined antigens and knowledge gaps in our understanding of M. avium subsp. paratuberculosis components eliciting pathogen-specific immune responses. We set out to (i) define a subset of proteins that contain putative antigenic targets and (ii) screen these antigen pools for immunogens relevant in detecting infection. To accomplish our first objective, we captured and resolved M. avium subsp. paratuberculosis-secreted proteins using a 2-step fractionation method and reverse-phase liquid chromatography to identify 162 unique proteins, of which 66 had not been previously observed in M. avium subsp. paratuberculosis culture filtrates. Subsequent screening of M. avium subsp. paratuberculosis-secreted proteins showed four antigens, of which one or more reacted on immunoblotting with individual serum samples from 35 M. avium subsp. paratuberculosis-infected cows. Moreover, these novel antigens reacted with sera from 6 low M. avium subsp. paratuberculosis shedders and 3 fecal-culture-positive cows labeled as ELISA seronegative. The specificity of these antigens was demonstrated using negative-control sera from uninfected calves (n ‫؍‬ 5) and uninfected cows (n ‫؍‬ 5), which did not react to any of these antigens in immunoblotting. As three of the four antigens are novel, their characterization and incorporation into an ELISA-based format will aid in detecting asymptomatic cattle in early or subclinical stages of disease. E nzyme-linked immunosorbent assays (ELISAs) are simple, rapid, and cost-effective tests that have been used for decades for determination of infection status. One of the major challenges in the development of an effective ELISA is the selection of antigens that are pathogen specific and permit sensitive detection. Antibodies against shared epitopes in closely related species can contribute to cross-reactivity (resulting in false-positive identification), and fluctuations in antibody titers and antibody compositions in chronic diseases hinder the development of sensitive tests. These factors have been problematic for the development of ELISAs for all mycobacterial diseases, including human tuberculosis (Mycobacterium tuberculosis), leprosy (Mycobacterium leprae), bovine tuberculosis (Mycobacterium bovis), and Johne's disease (Mycobacterium avium subsp. paratuberculosis).Johne's disease is a chronic gastroenteritis affecting ruminant animals worldwide, with national prevalence rates of 32% in Canada and 68% in the United States (1, 2). Calves are most susceptible and, once colonized, they remain asymptomatic for 2 to 5 years while shedd...

Identification of Novel Seroreactive Antigens in Johne's Disease Cattle by Using the Mycobacterium tuberculosis Protein Array

“…The M. avium subsp. paratuberculosis ortholog of this protein is MAP1569, which has been observed as a strong antigen in multiple serological screening approaches (12)(13)(14)(15). The second strongest antigen among the cows in the clinical stage of disease was Rv2878c, which is homologous to MAP2942c, another known antigen in M. avium subsp.…”

Characterization of the inflammatory phenotype of Mycobacterium avium subspecies paratuberculosis using a novel cell culture passage model

“…Unfortunately, mouse models offer limited insight on the complete disease progression, as mice do not exhibit intestinal inflammation in the same manner as ruminants in advanced stages of Johne's disease. MAP infections of goat and calf models are used to study a variety of disease interactions including host gene expression (Khare et al, 2012), vaccine efficacy (Hines et al, 2014), both bacterial (Facciuolo et al, 2013) and host (David et al, 2014) biomarker discovery for enhanced diagnostics, and are used to track and develop mathematical shedding and transmission models (Mitchell et al, 2008); however, the excessive time and space costs of large animals make ruminant models challenging to use. Though recent efforts have been made to standardize animal models used in Johne's disease research (Hines et al, 2007), historical literature lacks consistency.…”

“…At present, simple in vitro culture models of single-cell monolayers are used to explore the interaction of MAP with the epithelial intestinal cell or with phagocytic macrophage cells. Mouse models are typically employed to investigate initiation and prevention of infection during the early stages of Johne's disease Bermudez et al, 2010;Scandurra et al, 2010), while goat and calf models are more commonly used to study vaccine efficacy and the later stages of the disease (David et al, 2014;Facciuolo et al, 2013;Hines et al, 2014;Khare et al, 2012). While large animals provide useful models since they are the natural ruminant hosts of MAP and progress through the entire spectrum of disease states, they come with a variety of disadvantages.…”

Knowledge gaps that hamper prevention and control ofMycobacterium aviumsubspeciesparatuberculosisinfection

“…Analyses of longitudinal serum samples (from experimentally infected calves) were used to detect developing humoral immune responses against MAP proteins (Bannantine et al, 2008), with anti-MAP antibodies detected as early as 70 day after infection. Novel MAP antigens for detection of antibody responses in subclinical cattle were also reported (Facciuolo, Kelton, & Mutharia, 2013).…”