Fluorescence polarization assay for the detection of antibodies to Mycobacterium bovis in bovine sera

Abstract

The performance of a fluorescence polarization assay (FPA) that detects antibodies to Mycobacterium bovis in bovine sera is described. The FPA reported here is a direct binding primary screening assay using a small polypeptide derived from the M. bovis MPB70 protein. A secondary inhibition assay confirms suspect or presumed positive samples. Specificity studies involved five different veterinary laboratories testing 4461 presumed negative bovine samples. FPA specificity was 99.9%. The FPA was used to identify herd status as either M. bovis infected or non-infected. Herd surveillance studies (nine herds) were performed in Mexico and South Africa. The FPA had a specificity of 100% (two negative herds), and correctly identified six of seven infected herds. Finally, sera from 105 slaughter animals that had gross lesions in lymph nodes similar to those seen with bovine tuberculosis were tested by the FPA. Thin sections from the associated formalin-fixed paraffin-embedded samples of lymph nodes were stained using hematoxylin and eosin (H&E) for morphologic examination and using the Ziehl–Neelsen (ZN) method for detection of acid-fast bacilli. Of the 105 animals, 78 were classified as TB suspect based on lesion morphology, 21 were positive by ZN, 9 were positive by FPA and 13 were positive by PCR for the tuberculosis group of Mycobacterium. Among the 21 ZN positives, 11 (52.4%) were PCR positive. Among the 9 FPA positives, 8 (88.9%) were PCR positive. For the 13 PCR positives, 8 (61.5%) were FPA positive and 11 (84.6%) were ZN positives. These results show that use of the FPA for detection of M. bovis infection of cattle has value for bovine disease surveillance programs.

Introduction

M. bovis is the cause of bovine tuberculosis, an important disease that affects animal health and the economic value of cattle and food products derived from cattle. Furthermore, this disease is transmissible to other species of animals and humans. Bovine tuberculosis can progress insidiously through a herd of cattle without any obvious early stage clinical indications.

Veterinarians rely on several diagnostic methods to detect M. bovis infection (De la Rua-Domenech et al., 2006). The M. bovis surveillance testing is performed on cattle at strategic control points such as before international transport, breeding services, sale or exchange, and carcass inspections at slaughterhouses. The various diagnostic methods used may be well suited for one stage of the disease progression but not necessarily others. Each diagnostic method or point of surveillance offers some advantage but has associated disadvantages.

The most frequently used diagnostic methods for bovine tuberculosis rely on the cell mediated immune (CMI) response to test for exposure to M. bovis. Diagnostic tests for CMI include the caudal fold test (CFT), the comparative cervical test (CCT), and the gamma interferon test (GIT). CMI tests offer the advantage of early detection of tuberculosis, as the CMI response against M. bovis is more readily detected in the initial stages of the disease than is the humoral immune response. A disadvantage of diagnostic tests that are based on the CMI response is specificity, as false positive tests occur in cattle exposed with organisms related to M. bovis (Hope et al., 2005). In addition to problems with specificity, the CMI diagnostic methods have a relatively high total cost per test. The CFT and CCT require at least two visits to the farm by an accredited veterinarian with a 3-day interval between visits. Further, cattle must be restrained for each visit; interpretation of the results is subjective; and tuberculin materials vary in formulation throughout the world, raising the issue of comparability or equivalency of testing results. For the GIT, there is a relatively high expense for the test kit, and sample handling requirements are demanding. The harvested animal blood must be transported to a central laboratory within 30 h for further processing, and skilled technicians are needed to conduct the test.

For all three of the diagnostic methods that rely on the CMI response there is the risk that as M. bovis disease advances, a severely infected animal may become anergic and fail to mount a detectable CMI response (Ritacco et al., 1991, Surujballi et al., 2002). Thus, conventional CMI methods may fail to detect infected animals with long standing M. bovis infection.

Another important diagnostic method for detection of tuberculosis is histopathologic examination conducted post mortem. This requires considerable technical skill, an equipped histopathology laboratory, and substantial time to perform. This method is not well suited to large volume surveillance testing and is limited in that definitive identification of M. bovis is not possible. Consequently, animals and herds may be identified incorrectly as infected with M. bovis if other tests are not used for diagnostic confirmation. This false diagnostic result could lead to economic losses for producers.

The diagnostic “gold standard” methods for M. bovis detection are culture of the organism from affected tissues followed by confirmation of identity of the organism by PCR or, in some cases, PCR from fresh tissue. Culture offers isolation of the organism for further testing to achieve unambiguous identification of M. bovis, while PCR identifies the Mycobacterium tuberculosis complex that includes M. bovis. Both methods are more specific than CMI assays or histopathologic methods. However, a serious limitation of both culture and PCR is that they can only be performed reliably on samples collected post mortem. Culture of M. bovis requires stringent sample handling and advanced technical skill. Furthermore, culture methods can require 4 months for results. PCR requires sophisticated laboratory equipment, facilities, and advanced technical skills. Generally, a sample found to be positive by either culture or PCR is regarded as a true positive. However, a negative result by either method means that M. bovis was not detected in the sample of tissue that was tested. Other samples of tissue from the same animal could harbor M. bovis. Therefore, a negative test result from either culture or PCR does not provide conclusive proof that an animal is not infected with M. bovis, especially if the animal originated from an infected or suspect herd. While use of the “gold standard” methods is critical for confirmation of diagnosis, those methods are not well suited for large volume surveillance, where ease of use, assay speed, and large volume testing capabilities are important.

Serological assays provide an important and needed tool for large volume testing for exposure to M. bovis. They offer the important advantages of ease of use, assay speed and relatively low cost. A serological assay for M. bovis testing would complement the other established M. bovis diagnostic methods and facilitate diagnosis of the disease (Lin et al., 1996, De la Rua-Domenech et al., 2006).

There have been several reports of the development of low cost serological tests that might provide a more definitive diagnosis of M. bovis infection (Wood et al., 1992, Wood and Rother, 1994, Lin et al., 1996, Surujballi et al., 2002). A new antibody-based test has recently been described which offers the potential for the early detection of M. bovis infection in cattle (Waters et al., 2006).

Fluorescence polarization assay (FPA) detects the binding of a fluorescent low molecular weight moiety (tracer) to its high molecular weight binding partner by determining the tracer's fluorescence polarization (FP). When bound (the binding partner is present), the tracer exhibits a high FP; when free (the binding partner is absent), the tracer has a low FP (Nasir and Jolley, 1999). FPA has been applied to serological immunodiagnostics for various veterinary diseases (Jolley and Nasir, 2003, Nasir and Jolley, 1999). FPA is well suited for animal disease general surveillance because of its rapidity, ease of use and high sensitivity and specificity (Jolley and Nasir, 2003, Gall and Nielsen, 2004). As a homogeneous assay, FPA is a quantitative method performed either in a single tube or in a microtiter plate format, with short incubation times and no washing or separation steps. FPA is being used for surveillance as part of Brucellosis Eradication and Control programs following recent United States Department of Agriculture (USDA) and World Organisation for Animal Health (OIE) approvals and European Union pending regulatory review (Nielsen et al., 1996, Nielsen et al., 1998, Nielsen et al., 2004, Nielsen and Gall, 2001, Gall and Nielsen, 2004).

The MPB70 protein, secreted by M. bovis and other members of the M. tuberculosis complex, is a major immunodominant antigen (Wood et al., 1988, Lin et al., 1996). This protein is not present in Mycobacterium avium subsp. avium or Mycobacterium avium subsp. paratuberculosis which are other members of the genus having epidemiological importance. A FPA for the detection of antibodies to M. bovis using the whole MPB70 protein has been described (Lin et al., 1996, Surujballi et al., 2002). Here we describe the use of a FPA employing a polypeptide-based tracer, derived from the whole MPB70 protein (Jolley and Nasir, 2003) as a diagnostic tool for M. bovis surveillance in bovine populations. The peptide sequence corresponds to certain amino acids of the MPB70 protein (amino acids 51–78), showing the most reactivity with control sera from positive cattle, with a C-terminal lysine added to improve solubility. The tracer (F-733) was peptide 733 N-terminally labeled with 6-carboxyfluorescein.

The study was organized in three parts: (a) FPA general specificity studies carried out in the United States, Mexico, and South Africa; (b) FPA herd classification studies performed in Mexico and South Africa; (c) a Mexican slaughterhouse survey of carcasses that compared results of FPA on serum with results of testing lymph nodes with lesions, using acid-fast staining and PCR.