Use of Synthetic Peptides Derived from the Antigens ESAT-6 and CFP-10 for Differential Diagnosis of Bovine Tuberculosis in Cattle

Abstract

In Great Britain an independent scientific review for the government has concluded that the development of a cattle vaccine against Mycobacterium bovis infection holds the best long-term prospect for tuberculosis control in British herds. A precondition for vaccination is the development of a complementary diagnostic test to differentiate between vaccinated animals and those infected with M. bovis so that testing and slaughter-based control strategies can continue alongside vaccination. To date bacillus Calmette-Guérin (BCG), an attenuated strain of M. bovis, is the only available vaccine for the prevention of tuberculosis. However, tests based on tuberculin purified protein derivative cannot distinguish between M. bovis infection and BCG vaccination. Therefore, specific antigens expressed by M. bovis but absent from BCG constitute prime candidates for differential diagnostic reagents. Recently, two such antigens, ESAT-6 and CFP-10, have been reported to be promising candidates as diagnostic reagents for the detection of M. bovis infection in cattle. Here we report the identification of promiscuous peptides of CFP-10 that were recognized by M. bovis-infected cattle. Five of these peptides were formulated into a peptide cocktail together with five peptides derived from ESAT-6. Using this peptide cocktail in T-cell assays, M. bovis-infected animals were detected, while BCG-vaccinated or Mycobacterium avium-sensitized animals did not respond. The sensitivity of the peptide cocktail as an antigen in a whole-blood gamma interferon assay was determined using naturally infected field reactor cattle, and the specificity was determined using blood from BCG-vaccinated and noninfected, nonvaccinated animals. The sensitivity of the assay in cattle with confirmed tuberculosis was found to be 77.9%, with a specificity of 100% in BCG-vaccinated or nonvaccinated animals. This compares favorably with the specificity of tuberculin when tested in noninfected or vaccinated animals. In summary, our results demonstrate that this peptide cocktail can discriminate between M. bovis infection and BCG vaccination with a high degree of sensitivity and specificity.Bovine tuberculosis (BTB) is caused by Mycobacterium bovis. It is a zoonotic disease and was the cause of approximately 6% of total human deaths due to BTB in the 1930s and 1940s and of more than 50% of all cervical lymphadenitis cases in children (13, 23). The introduction of pasteurization of milk in the 1930s dramatically reduced the transmission from cattle to humans, and in 1995 only 1% of 3,200 isolates from patients with TB in Great Britain were identified as M. bovis (23). However, human TB caused by M. bovis is still a major health issue in many developing countries (12,14,15).BTB has severe implications for animal welfare in both developed and developing countries, since it causes reduced productivity and premature death in cattle and since affected farms also suffer severe economic losses. A compulsory eradication program based on the slaughter of infected animals detec...

Identification of proteins from tuberculin purified protein derivative (PPD) by LC-MS/MS

“…Although whole-blood gamma interferon assays have been developed for diagnostic of bovine tuberculosis 32,33 , and depending on the antigens used, these tests can effectively differentiate vaccinated from infected animals 13,14,16 A key aim of this work was to identify proteins that could be used to generate an immunological reagent that could be used to differentiate M.…”

“…We detected four such antigens in bovine PPD but all of them (MPT64, CFP21, CFP10 and ESAT-6) have previously been investigated as antigens for development of diagnostic tests 13,16,42 capable of differentiating BCG-vaccinated animals from those infected with bovine tuberculosis [13][14][15][16] . Although no novel BCG-absent antigens were detected in the current analysis the fact that the study did pick up nearly all the known M. bovis-specific antigens demonstrates the power of this approach.…”

“…The recombinant proteins MPB59, MPB64, MPB70 and ESAT 6 were evaluated in a differential diagnostic test, but only ESAT 6 was shown to be suitable for differentiating BCG-vaccinated animals from those infected with bovine tuberculosis 13,14 . Synthetic peptides derived from ESAT6, CFP-10, MPB64, MPB70 and MPB83 were tested alone or in combination, with limited success 15,16 .…”

Comparative Analysis of B- and T-Cell Epitopes ofMycobacterium lepraeandMycobacterium tuberculosisCulture Filtrate Protein 10

“…Both ESAT-6 and CFP-10 have shown great promise as immunodiagnostic reagents for TB in humans and cattle (2,5,39,44). It has been shown that combining M. tuberculosis ESAT-6 and CFP-10 in a diagnostic test for infection or exposure to TB could provide the level of specificity and sensitivity necessary to detect exposure to TB (2).…”

Identification of Mycobacterium bovis antigens by analysis of bovine T-cell responses after infection with a virulent strain

“…In an attempt to identify individual antigens in antigenic fractions using a combination of methods, we determined that these stimulant fractions consisted of 85B, TRB-B, MPB70, TPX, CFP10 and ESAT6. 85B (22), ESAT6 (6,23) and MPB70 (24)(25)(26) are well known T-cell-stimulating antigens, while the antigenicity of CFP10 has only recently been demonstrated (23,25,27). These results support the view that low molecular weight proteins such as ESAT6 (6) play an important role in bovine immune responses to M. bovis.…”