Short communicationEvaluation of two cocktails containing ESAT-6, CFP-10 and Rv-3615c in the intradermal test and the interferon-γ assay for diagnosis of bovine tuberculosis
Introduction
Eradication programs of bovine tuberculosis (Mycobacterium bovis) are based on prompt detection of infected animals and their removal from the herd. Diagnostic tests used routinely are based on cell-mediated immunity (CMI) response (McNair et al., 2007): intradermal tuberculin tests and the interferon-gamma (IFN-γ) assay (Rothel et al., 1992). In their original form, these tests are based on the response against purified protein derivative (PPD) tuberculin. Bovine PPD is a poorly defined mix of proteins, lipids and carbohydrates obtained from M. bovis culture AN5 (OIE, 2011) and it lacks specificity because some of its antigenic components are present in non-pathogenic environmental mycobacteria (Dunn et al., 2005, Hope et al., 2005).
Defined antigens such as early secretory antigenic target-6 kDa (ESAT-6), culture filtrate protein 10 (CFP-10), or combination/s of them, have been extensively tested in the IFN-γ assay with satisfactory results (Vordermeier et al., 2001, Aagaard et al., 2010). A limited number of studies have shown the feasibility of the use of these antigens for the specific skin test in humans (Bergstedt et al., 2010) and in the guinea pig model (van Pinxteren et al., 2000, Weldingh and Andersen, 2008). The studies performed in cattle with rESAT-6 (Pollock et al., 2003, Whelan et al., 2003) and cocktails combinations of ESAT-6, CFP-10 and other antigens (Whelan et al., 2010) have also yielded promising results. However, information of its usefulness in field diagnosis of bovine tuberculosis remains scarce.
The target of the present study was to assess the application of definite reagents previously defined by Whelan et al. (2010) using both the single intradermal test and the IFN-γ assay in cattle naturally infected with M. bovis and to compare their performance with the application of the standard bovine PPD.
Section snippets
Herd and design of the study
The study was performed in cattle from a herd with chronic natural M. bovis infection. Cattle (n = 23) were simultaneously subjected to SIT test using the different antigens (bovine PPD, peptide and protein cocktails) and were tested in parallel with the in vitro IFN-γ assay. Handling of the animals, testing and sampling were performed by veterinarians in accordance with institutional guidelines according to Spanish Legislation.
Antigens
Bovine PPD (1 mg/ml) was kindly provided by CZ Veterinaria (Porriño,
SIT test
The number of reactors varied depending on the antigen injected but these differences were not statistically significant (p > 0.05). Using the severe interpretation the number of reactors was 14/23 (60.9%, confidence interval 40.8–77.8) cattle when the test was performed with bovine PPD at 0.1 mg, 11/23 (47.8%, 95% CI 29.2–67) animals were classified as reactors when the test was performed with the peptide cocktail, and 14/23 (60.9%, CI 40.8–77.8) tested positive when the test was performed with
Discussion
Skin testing with PPD remains the first-line ante-mortem diagnosis technique to detect exposure to members of the M. tuberculosis complex in humans and animals. During the last decade, significant efforts have been devoted to the development of new specific antigens (Vordermeier et al., 2011) that have been widely evaluated in in vitro IFN-γ assay. Although two pilot studies (Pollock et al., 2003, Whelan et al., 2010) have provided the proof-of-principle that these antigens are applicable for
Acknowledgements
This study was funded by the EU Project FP7-KBBE-2007-1 “Strategies for the eradication of bovine tuberculosis (TB-STEP)”. C. Casal is recipient of a research contract assigned by Comunidad de Madrid (FINNOVA II Programme). We thank the Mycobacteria and Computer and Communication Units staff for their technical support.
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