Multiplex Immunoassay for Serological Diagnosis of Mycobacterium bovis Infection in Cattle

Abstract

Efforts to develop a better diagnostic assay for bovine tuberculosis have shown that the sensitivity and specificity of an assay can be improved by the use of two or more antigens. As reported here, we developed a multiplex chemiluminescent immunoassay that can simultaneously detect antibody activity to 25 antigens in a single well in a 96-well plate array format. The chemiluminescent signal is captured with a digital imaging system and analyzed with a macro program that tracks each serum for its pattern of antibody activity for Mycobacterium bovis antigens. The comparison of sera from 522 infected and 1,489 uninfected animals showed that a sensitivity of 93.1% and a specificity of 98.4% can be achieved with a combination of antigens. The assay system is rapid and can be automated for use in a centralized laboratory.Bovine tuberculosis (bTB) continues to be a zoonotic disease affecting multiple species, including humans (3, 9, 21, 26). The disease has been difficult to control in livestock because of the lack of an effective vaccine, the presence of wildlife reservoirs, and the lack of a diagnostic assay with sufficient sensitivity and specificity to detect animals at all stages of infection. Currently, the primary methods used for the detection of TB in humans and ruminants include the measurement of a delayedtype hypersensitivity (skin test) to purified protein derivative (PPD) and an indirect in vitro assay that measures the concentration of gamma interferon (IFN-␥) produced in response to stimulation with PPD (22,30,31). Although the methods have proven useful in controlling bTB, they lack sensitivity and specificity because of a cross-reactive immune response to Tand B-cell epitopes conserved on orthologous molecules present in nonpathogenic mycobacteria and Mycobacterium avium subsp. paratuberculosis (reviewed in references 8, 23, and 27). To obviate this problem, an extensive effort has been under way to identify and characterize antigens unique to Mycobacterium bovis that could be used in a diagnostic assay. To date, studies have shown that the antibody response to M. bovis is not uniform, with no evidence of a dominant persistent response to a single antigen (reviewed in references 4, 7, and 8) at any stage of infection (2,19). This finding has suggested that some type of a multiplex assay is needed to detect animals at different stages of infection (1, 2). However, the necessity of using multiple antigens in an assay has introduced another challenge. The evaluation of the standard type of enzymelinked immunosorbent assay (ELISA) has shown that sensitivity and specificity are reduced when multiple antigens are combined for analysis in a single well, thus limiting the way a conventional ELISA can be used (20). To address this problem, we developed a multiplex assay that can simultaneously detect and analyze the response to multiple antigens spotted in a single well in a 96-well plate array format. We demonstrate the enhanced diagnostic power of a multiplex antigen approach over that of the industry-stand...

Serological Analysis of Tuberculosis in Goats by Use of the Enferplex Caprine TB Multiplex Test

“…In previous work, we have described the development of multiplex serology tests by using antigen arrays to detect antibodies to specific M. bovis antigens in several species, including cattle, goats, and alpacas (15)(16)(17)(18)(19)(20)(21). The sensitivities and specificities obtained with these serology tests varied with the population under study.…”

Evaluation of a Commercial Enzyme-Linked Immunosorbent Assay for the Diagnosis of Bovine Tuberculosis from Milk Samples from Dairy Cows

“…More recently, the whole-blood gamma interferon (IFN-␥) test has been used in conjunction with the skin test to improve specificities by retesting skin test reactor cattle or to improve sensitivities by retesting skin test-negative animals from chronically infected herds (2, 3). Although the IFN-␥ test has made a valuable contribution in improving the accuracy of diagnosing TB in cattle, additional types of assays are needed, particularly assays which are inexpensive and which can potentially be used on pooled samples from groups of animals or identify animals not responding in cell-based assays.Serological assays have shown promise for diagnosing M. bovis infections in animals which have not responded in skin tests (4,5,6), particularly those which have severe pathology and are more likely to shed M. bovis. A serological assay developed to detect antibodies to M. bovis in milk samples provides advantages in that milk samples are routinely collected for dairy herd improvement testing and can be pooled from groups of animals.…”

“…Serological assays have shown promise for diagnosing M. bovis infections in animals which have not responded in skin tests (4,5,6), particularly those which have severe pathology and are more likely to shed M. bovis. A serological assay developed to detect antibodies to M. bovis in milk samples provides advantages in that milk samples are routinely collected for dairy herd improvement testing and can be pooled from groups of animals.…”

TUBERCULOSIS AND BRUCELLOSIS IN WOOD BISON (BISON BISON ATHABASCAE) IN NORTHERN CANADA: A RENEWED NEED TO DEVELOP OPTIONS FOR FUTURE MANAGEMENT

“…The caudal fold test is not sufficiently sensitive to detect all clinical cases of bTB in bison, and newly developed serologic tests may be more sensitive (Himsworth et al 2010a). Several serologic tests for bTB, including the Cervid TB StatPak TM (Chembio Diagnostics Ltd, Medford, New York, USA), EnferPlex TB Assay TM (Enfer Group, Kildare, Ireland), and IDEXX TB Assay TM (IDEXX Laboratories, Westbrook, Maine, USA), were developed for cattle and cervids (Lyashchenko et al 2000;Whelan et al 2008Whelan et al , 2010O'Brien et al 2009;Buddle et al 2010;Schiller et al 2010;Clegg et al 2011), but few, if any, are validated for bison. c-Interferon assays also have broad utility in cattle testing, but little work has been performed in bison (Schiller et al 2010), although these tests were useful in African buffalo (Syncerus caffer; Michel et al 2011).…”