Identification of Novel Mycobacterium bovis Antigens by Dissection of Crude Protein Fractions

Abstract

Culture filtrate and cell extracts from Mycobacterium bovis cultures contain molecules which could promote protective immunity to tuberculosis in animals. Different protein fractions of M. bovis cultures were obtained by elution electrophoresis and were tested in experimentally infected cattle. The fractions that elicited gamma interferon (IFN-␥) responses were resolved by two-dimensional gel electrophoresis, and individual proteins were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The open reading frames were cloned, expressed as their recombinant forms, and retested with naturally and experimentally infected animals. Eleven protein fractions were highly reactive, from which the Rv1636, HspX, Rv0138, Rv2524, EsxI, and Rv3740 recombinant proteins were obtained. EsxI and HspX were the antigens most recognized by the IFN-␥ release assay. In summary, a proteomic approach allowed the identification of novel antigens useful for the diagnosis of bovine tuberculosis.Infection and disease with Mycobacterium bovis, the causative organism of bovine tuberculosis (TB), is an important health problem in cattle and other animal species. The disease presents a major barrier to animal-related trade and production, causing significant losses to farming economies worldwide. Furthermore, bovine TB has serious zoonotic implications, especially in developing regions. In North and South America, there are 420 million cattle, half of which are in countries with an incidence of bovine TB higher than 1% (13). The disease control programs carried out in most countries are based on a test and removal strategy utilizing the tuberculin skin test (TST) with purified protein derivative (PPD) (4). Accurate detection using the TST and removal of infected cattle form the base of control strategies for bovine TB. It is widely recognized that tests for cell-mediated immunity are far more sensitive than antibody tests in detecting bovine TB (38). Disease control based on the use of PPD can be facilitated by using the gamma interferon (IFN-␥) test for bovine TB (39). The PPDs are obtained by acid precipitation from heat-killed cultures of M. bovis (PPD-B) and Mycobacterium avium (PPD-A) and are poorly defined (27), containing many proteins, lipids, and sugars. Many of these compounds are shared by different mycobacterial species or even with other bacteria (22). The detection of false positives has been a significant part of the economic cost, particularly in areas where there is a relatively low incidence of the disease (28), and thus, there is an urgent need for more specific reagents for the diagnosis of M. bovis infection.Many mycobacterial proteins have been isolated, and bovine T-cell epitopes have been characterized for several of them (2, 9, 26, 30, 32). Immunodominant peptides from M. tuberculosis Rv3873, Rv3879c, Rv3019c, Rv0288, ESAT-6, and CFP-10 genes were described as good T-cell inducers, and the diagnostic potential of a cocktail composed of these dominant peptides was demonstrated using...

Evaluation of the use of recombinant proteins of Mycobacterium bovis as antigens in intradermal tests for diagnosis of bovine tuberculosis

“…On the other hand, PE5 is a member of the PE family that is present in the mycobacterial cell wall, and is considered, along with TB10.4 and PE13, to be an antigen that is sensitive and highly specific in the IFN-γ assay (Aagaard et al 2006). Although Mb0143 was also considered a satisfactory antigen in IFN-γ assay to identify experimentally infected cattle (Meikle et al 2009), its sensitivity levels were not satisfactory in the skin test.…”

“…In the present study, proteins, such as EsxI (Jones et al 2010), Mb0143, PE5, PE13 and TB10.4 previously assessed in IFN-γ assays (Aagaard et al 2006, Meikle et al 2009, were evaluated in skin test in cattle. Excluding TB10.4, which was recently evaluated in skin test in cattle by Xin et al (2013), all the other proteins were assessed for the first time in skin test in bovines.…”

“…Based on previous reports of diagnosis of tuberculosis in cattle using IFN-γ assays (EsxI, Mb0143, PE5, PE13 and TB10.4) (Aagaard et al 2006, Jones et al 2010, Meikle et al 2009, the aim of the present study was to assess these recombinant proteins with respect to the ability to differentiate M. bovis sensitised and non-sensitised cattle by skin test, for further evaluation of the best antigens in infected animals.…”

Improving the characteristics of a mycobacterial 16 kDa-specific chicken scFv

“…Together with ELISA these results have shown that random mutagenesis and shortening the linker can improve the binding characteristics of chicken scFvs. Since the 16 kDa TB protein is known to be a useful diagnostic target for serodiagnosis and cellular immunoassays [5,6], the engineered scFvs described here are likely to be useful research tools and in diagnostics development.…”

“…This antigen is considered to be a potentially useful diagnostic target for serodiagnosis of TB in humans [5] and can also be used to stimulate lymphocytes in the gamma interferon test for bovine TB [6]. Antibodies directed against this protein are therefore likely to be useful in research and diagnosis.…”