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Evaluation 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

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Abstract

The intradermal tuberculin tests and the interferon-gamma (IFN-γ) assay are the principal tests used worldwide for the ante-mortem diagnosis of bovine tuberculosis. The conventional reagent currently in use in these tests is purified protein derivative (PPD) tuberculin obtained from Mycobacterium bovis culture. The components of PPD are poorly characterized and difficult to standardize. To overcome this issue, antigens specific to the Mycobacterium tuberculosis complex are being studied. Here we have assessed the biological potency of ESAT-6, CFP-10 and Rv-3615c presented as peptide or recombinant protein cocktails in comparison with the standard bovine PPD used routinely in Spanish eradication campaigns. The study was performed in cattle (n = 23) from a herd with natural M. bovis infection. Animals were simultaneously injected with PPD and the peptide and protein cocktails. The percentages of cattle reacting positively to single intradermal test were 60.9% (bovine PPD), 47.8% (peptide cocktail) and 60.9% (protein cocktail), with no significant difference between the actual skin fold thickness increases (p > 0.05). The IFN-γ assay detected 60.9% of animals when stimulation was performed with bovine PPD, but decreased to 52.2% when stimulation was performed with the peptide cocktail and to 47.8% when stimulation was performed with the protein cocktail. However, no significant differences were found between IFN-γ responder frequencies (p > 0.05). These results show a potential use of these defined reagents for in vivo tuberculosis diagnosis.

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.

References (19)

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