Introduction

Tuberculosis (TB) is a leading cause of morbidity and mortality in the world, also affecting a wide range of animal species, particularly livestock, in both developed and developing countries. The disease is caused by tuberculous mycobacteria belonging to the Mycobacterium tuberculosis complex (MTC). This complex consists of several closely-related pathogenic species, namely M. tuberculosis, the main agent of human TB, and, amongst others, M. bovis and M. caprae that are primary agents of bovine and caprine TB, respectively [1]. These species are genetically very similar but may differ in host preference and epidemiological characteristics [2]. Mycobacterium bovis and M. caprae also represent a high potential for zoonotic transmission to humans [3–5], with evidence of possible person-to-person transmission [6]. However, the main routes of transmission are the contact with infected animals and ingestion of unpasteurized dairy products. These zoonotic MTC species may be responsible for up to 7.2% and 15% of human TB cases in industrialized and developing countries, respectively [7]. Rapid and reliable laboratory tests for the direct detection of tuberculous mycobacteria in biological samples are in high demand, in both human health and veterinary settings, and are crucial for an improved TB control.

In most developed countries, bovine tuberculosis has been tackled during the last decades by costly eradication programs, involving the culling of reactor animals and laboratory testing of suspect samples for the definitive confirmation of the presence of MTC. Presently, the detection of MTC bacteria in animal tissues is mainly based in lengthy and cumbersome conventional methods, involving the examination of Ziehl-Neelsen stained smears, histopathology and culture in selective media, followed by biochemical or molecular identification of typical mycobacteria colonies. The microscopic identification of acid-fast mycobacteria is non-specific and highly insensitive, particularly in the case of paucibacillary forms of TB. Culture remains the gold-standard method to confirm TB infection but requires several weeks to obtain positive results due to the extremely fastidious growth of tuberculous mycobacteria. In spite of the significant advances in the development of novel molecular diagnostic assays towards a faster and accurate detection of MTC in human samples, only a few assays have been described for detecting these agents in animal tissues, particularly in fresh tissues from livestock [8-12]. Most of these molecular approaches are PCR-based and target specific polymorphisms, insertion sequences, and the so-called regions of difference in the genome of MTC members [9,10,13-18]. Nevertheless, most of the amplification-based assays described for detecting MTC nucleic acids directly in fresh or formalin-fixed paraffin-embedded tissues only yield a moderate sensitivity, usually up to 75%, particularly when testing tissues without the characteristic lesions or detectable acid-fast bacilli [8-10,15,19]. This limitation is mostly related to the inefficiency of mycobacterial DNA extraction procedures from affected animal tissues, especially those exhibiting strong fibrosis, calcification, and with no histological evidence of acid-fast bacteria [8,19]. The use of immunomagnetic separation approaches to concentrate mycobacteria cells from animal tissues prior to DNA extraction may enhance PCR sensitivities [20,21]. Nevertheless, these approaches usually involve more experimental steps and expensive equipment and consumables not readily available in veterinary diagnostic settings.

In the present work we have developed a novel and simple Taqman-based semi-nested real-time PCR assay yielding extremely high sensitivity and specificity for the direct detection of tuberculous mycobacteria in fresh animal tissues, namely of bovine origin, capable of being introduced in routine diagnostic veterinary laboratories.

Materials and Methods

Design of TaqMan probes and flanking primers

Sequences of IS6110 from MTC members and β-actin gene from a wide range of mammal species were retrieved from NCBI-GenBank. Comparative analysis of these two sets of sequences was achieved through sequence alignment using the CLUSTAL X v2.0 software [29]. Complementary regions for a β-actin gene-targeted mammals-universal TaqMan probe and flanking degenerated primers were found after visual inspection of the respective alignments (Table 3; Figure 1). The amplification of β-actin gene served as control to detect inhibition of the PCR reactions when using DNA extracted from tissues as template. The IS6110-targeted probe (P_IS6110) and respective flanking primers (F_IS6110 and R_IS6110) were retrieved from the study of Restrepo and colleagues [30] (Table 3; Figure 2). An additional IS6110-targeted forward primer (FN_IS6110) for use in a semi-nested PCR was designed (Table 3; Figure 2). Probes and primers specificities were tested in silico using the BLAST tools from NCBI-GenBank. All probes and primers targeting IS6110 and β-actin gene were synthesized by MWG Biotech (Germany).

Primer/Probe	Sequence (5´-3´)	Complementary target
F_Actin	GGC TCY ATY CTG GCC TC	β-actin gene of mammals
R_Actin	GCA YTT GCG GTG SAC RAT G
P_Actin1	Cy5.5-TAC TCC TGC TTG CTG ATC CAC ATC-BHQ2
F_IS6110	GGG TCG CTT CCA CGA TG	IS6110 element of MTC species
FN_IS6110	CTC GTC CAG CGC CGC TTC GG
R_IS6110	GGG TCC AGA TGG CTT GC
P_IS6110 2	FAM-CGC GTC GAG GAC CAT GGA GGT-BHQ1

Table 3. Sequences of primers and probes used in this study.

¹Probe labeled with Cy5.5 fluorophore and BHQ-2 quencher; ²Probe labeled with carboxyfluorescein (FAM) fluorophore and BHQ-1 quencher

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Figure 1. Complementary targets of the mammals β-actin gene targeted probe and flanking primers.

Partial alignment of β-actin gene sequences of several mammal species showing the complementary targets of the P_Actin TaqMan probe and respective flanking degenerated primers (F_Actin and R_Actin) (gray boxes). The GenBank access numbers from which the partial sequences were retrieved are indicated for each species inside parenthesis. Mismatches in relation to consensus sequence are highlighted in red.

https://doi.org/10.1371/journal.pone.0081337.g001

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Figure 2. Complementary targets of the MTC-specific IS6110-targeted probe and flanking primers.

Partial alignment of the IS6110 sequence of Mycobacterium tuberculosis complex members with other IS6110-like sequence recently found in M. smegmatis (GenBank access numbers are indicated inside parenthesis). The complementary targets of the P_IS6110 TaqMan probe and flanking primers (FN_IS6110, F_IS6110 and R_IS6110) are indicated (gray boxes). Mismatches in sequences are highlighted in red.

https://doi.org/10.1371/journal.pone.0081337.g002

Results

Design of probes and primers

A novel set of β-actin gene-targeted TaqMan probe and respective flanking primers was designed (Table 3, Figure 1). In silico analysis using the BLAST suite of NCBI-GenBank confirmed that the complementary regions of this probe, and primers, are widespread amongst most mammal species, including livestock animals. In silico analysis also confirmed that the complementary regions of the IS6110-targeted probes and primers were only present in IS6110 sequences of MTC members (Table 3, Figure 2).

Analytical specificity and sensitivity.

The real-time PCR assay with the IS6110-targeted probe (P_IS6110) and flanking primers (F_IS6110 and R_IS6110) yielded amplification products only when using DNA extracted from MTC members as template (Table 1, Figure 3). No non-specific results were obtained with members of the Mycobacterium avium complex or with strains belonging to other diverse bacterial species. The minimum detection threshold (analytical sensitivity) of this assay was estimated by the construction of a reference curve with serially-diluted suspensions of DNA from M. tuberculosis H37Ra. The analytical sensitivity was estimated in 0.3 fg/µL of M. tuberculosis genomic DNA. A linear relationship between the logarithm of the starting concentration of DNA and the amplification Ct values was obtained, with a -3.222 slope, a Ct = 35 interception in the minimum threshold (0.3 fg/µL) and an R² = 0.999.

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Figure 3. IS6110-targeted real-time PCR amplification curves obtained in specificity tests.

Illustration of the MTC-specific amplification curves obtained in FAM channel during evaluation of the analytical specificity of the IS6110-targeted real-time PCR step. All MTC cultures yielded amplification curves: M. tuberculosis ATCC 25177 (blue line), M. bovis LNIV 13027 (green line), M. bovis ATCC 27291 (orange line) and M. caprae LNIV 17320 (red line). Other bacteria yielded negative results of amplification: M. avium subsp. avium ATCC 25291 (yellow line) and M. avium subsp. paratuberculosis LNIV 39888 (brown line). No amplification was detected in non-template negative control (black line). RFU - Relative Fluorescence Units.

https://doi.org/10.1371/journal.pone.0081337.g003

Spiked samples.

Tissue samples spiked with serially-diluted suspensions of M. tuberculosis cells were used for assessing the detection limit of the β-actin and IS6110-targeted semi-nested duplex real-time PCR assay. The detection threshold was estimated to be one mycobacteria per ml of tissue macerate (Figure 4). The IS6110 amplification curves usually harboured a very low Ct (

< 3), meaning that the first standard PCR step yielded abundant amplification products easily detected by the following real time PCR step . The co-amplification of the bovine β-actin gene showed that no apparent inhibition of the real-time PCR step occurred due to the presence of inhibitor components of tissues or excess of bovine DNA, a significant result for the type of specimens used (animal tissues), confirming the efficiency of the extraction and purification procedures used in this work (Figure 4).

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Figure 4. Testing of spiked samples with the semi-nested duplex real-time PCR assay.

The analytical sensitivity of the assay was estimated through the use of solutions of DNA extracted from MTC-free tissue samples spiked with serially diluted cellular suspensions of M. tuberculosis ATCC 25177 (ranging from 10⁷ to 10° cells/ml of tissue homogenate). The figure illustrates the IS6110 (solid lines) and mammal β-actin gene (dashed lines) targeted amplification curves obtained in FAM and Cy5.5 channels, respectively, for the several dilutions: 10⁷ (yellow line), 10⁵ (black line), 10³ (brown line), 10¹ (green line) and 10° (blue line) cells. Positive control: M. tuberculosis ATCC 25177 culture (red line); Negative controls: unspiked tissue homogenate (orange line) and non-template control (grey line). RFU - Relative Fluorescence Units.

https://doi.org/10.1371/journal.pone.0081337.g004

Diagnostic specificity and sensitivity.

The results of the semi-nested duplex real-time PCR assays using DNA extracted from tissue samples are summarized in Table 2. All but one tissue sample from which M. bovis was cultured (types I and II) yielded positive amplification results, corresponding to a diagnostic sensitivity of 98.2% (CI_P95% 89.4-99.9%). Noteworthy, when a subset of these TB culture positive samples was preliminary tested using only the second step of the amplification assay (without the first conventional PCR step), the diagnostic sensitivity was only 40%. Eight DNA-positive tissues were obtained among the 71 M. bovis culture negative samples (types III, IV, V and VI), yielding a diagnostic specificity of 88.7% (CI_P95% 78.5-94.7%). Negative controls run with batches of samples did not identify any cross-contaminating DNA. The positive and negative predictive values were estimated in 87.5% (CI_P95% 76.3-94.1%) and 98.4% (CI_P95% 90.1-99.9%), respectively, for a prevalence of 44.5% M. bovis culture positive samples among all samples analysed. Considering only the bovine samples, the diagnostic sensitivity and specificity of the amplification assay were estimated in 100% (CI_P95% 84.0-100%) and 97.7% (CI_P95% 86.2-99.9%), respectively, while PPV and NPV were estimated in 96.3% (CI_P95% 79.1-99.8%) and 100% (CI_P95% 89.6-100%), respectively, for a prevalence of 37.7% M. bovis culture positive samples.

No DNA-positive samples were obtained from samples without TB-compatible lesions and M. bovis isolation (types III and IV) (Table 2). However, eight DNA-positive samples were detected among tissues that were M. bovis culture-negative but that harboured TB-compatible lesions (sample types V and VI) (Table 2).

An observed kappa coefficient was estimated in 0.859 (CI_P95% 0.771-0.948) for the overall agreement between the results obtained by the direct application of the IS6110-targeted semi-nested PCR assay to fresh tissue samples and the results obtained from the gold-standard bacteriological culture. The direct detection procedure allowed the detection of MTC infected samples in less than 6 hours while the conventional culture takes about 6 to 12 weeks.

Discussion

The availability of TB confirmatory tests allowing a fast and conclusive detection of tuberculous mycobacteria in suspect animal tissues would be a great advantage in improving the efficiency of TB eradication programs and in decreasing the associated economic burden. Improvements in specificity, sensitivity and detection limit of diagnostic assays are usually introduced by molecular approaches. Nevertheless, only a few diagnostic PCR-based assays have been described for detecting MTC members directly in animal specimens, usually yielding limited sensitivities when compared to the reference bacteriological culture [8-12,15,19]. These moderate sensitivities may well be linked to the increased complexity for disrupting and recovering genomic DNA from the tough mycobacterial cells and to the paucibacillary nature of many animal tissue lesions used for nucleic acids extraction [10]. The presence of amplification inhibitors in crude tissue extracts, namely of large amounts of co-extracted eukaryotic DNA, may represent an additional problem. An option that has been explored to enhance the sensitivity of PCR techniques involves the implementation of more effective DNA extraction and purification methods. Taylor and colleagues were able to increase the M. bovis PCR detection sensitivity in bovine tissue samples with visible lesions, from 70% to 91%, after the inclusion of an additional step of liquid nitrogen freeze-thaw cycles in the DNA extraction procedure [10]. The use of sequence capture or immunomagnetic separation approaches was also forecasted for recovering higher yields of mycobacterial DNA from samples [8,9,12,21]. Nevertheless, these DNA extraction approaches usually involve more experimental steps and expensive equipments and consumables.

In this work, an alternative approach was evaluated for enhancing MTC detection sensitivities directly from fresh animal tissues. An adapted and optimized user-friendly DNA extraction protocol, mainly based in the use of simple commercially obtainable extraction kits, was coupled with an IS6110-targeted semi-nested real-time PCR assay that allows the direct detection of MTC members in animal tissue specimens with very high sensitivity, specificity and positive and negative predictive values, namely in bovine specimens. These two last parameters are dependent on the prevalence of the tested condition in the population under study, as well as on the sensitivity and specificity of the testing assays. The prevalence of MTC (mostly M. bovis) infected samples among all TB-suspect samples submitted to the reference laboratory during the 2002-2010 period (n = 6364) was approximately 40% (reviewed by Cunha and colleagues [27]), which compares well with the prevalence values found in this work and used for the computation of PPV and NPV. Therefore, the PPV and NPV parameters computed may be considered good indicators of the performance of our IS6110-targeted assay for assessing the presence of MTC in specimens collected from animals clinically suspected of having tuberculosis and submitted for confirmatory culture analysis in a major reference laboratory.

Eight DNA-positive amplification results were obtained from tissue samples from which M. bovis could not be cultured (Table 2). However, lesions compatible with TB were observed in these tissues during histological analysis (sample types V and VI) (Table 2). The culture of tissue samples for the isolation of M. bovis and of other MTC members, followed by molecular or biochemical identification procedures, is usually the gold-standard method to validate alternative diagnostic assays. However, it is known that bacteriological culture is slow and laborious and can yield ambiguous or false-negative results, e.g. due to the presence of non-viable mycobacteria, raising concerns about its effectiveness as comparison reference method [19,31]. The results of culture assays may be affected by several factors, such as the harsh processing and decontamination procedures of samples, which can also have a harmful effect on M. bovis viability, as well as the growth media and incubation conditions used and the constrained distribution of mycobacteria in tissues [32]. Therefore, although TB-like lesions identified by histopathology can be induced by other bacteria or mycobacteria, the positive amplification results suggest that MTC members were most probably associated with the observed lesions and that the PCR test is more sensitive than bacteriological culture for detecting these pathogens in animal tissues. Non-MTC mycobacteria such as M. avium and M. scrofulaceum were cultured from five of those eight DNA-positive tissue samples (Table 2). These non-MTC mycobacteria may potentially overgrow any M. bovis isolate present in the sample and mask its presence [19]. Regardless of some discrepancies, the kappa measure of agreement between bacteriological culture and the semi-nested PCR was estimated in 0.859. Although the criteria for judging kappa statistic are not completely objective nor universally accepted, this value may allow us to infer an "almost perfect" agreement between the two MTC detection methods [33].

The enhancement of the MTC detection rates using the semi-nested amplification assay need to be balanced against the associated increased risk of cross contamination of samples. Therefore, we should emphasize the need of working in a veterinary diagnosis laboratory harboring good practice standard conditions for molecular analysis, which include working in separate clean rooms and the use of positive and negative controls.

In a preliminary survey the testing of TB positive and negative tissue samples employed a standard real-time PCR assay (corresponding to the second step of the semi-nested approach). The comparison of this assay with the reference of bacteriological culture showed a diagnostic sensitivity of only 40% (data not shown). The inclusion of the first step of conventional IS6110-targeted PCR amplification, in a semi-nested design, allowed to increase the sensitivity of the assay to near 100%. Previous studies found no significant improvements in the detection of MTC members in animal tissue samples using nested PCR assays, including with real-time PCR formats [10,14,15]. Nevertheless, the performance of PCR detection systems is highly dependent on the efficiency of the primers and probes, even when using the same genomic targets such as the IS6110 element [34]. The MTC-specific IS6110-targeted primers and probe used in this work were shown to be highly efficient for detecting tuberculous mycobacteria in animal tissue samples, although the isolates of the most relevant species, M. bovis, usually contain only one copy of this insertion sequence. It has recently been found that IS6110-like elements may be present in other non-MTC mycobacteria such as M. smegmatis [35]. However, the probe and respective flanking primers used show no relevant complementary regions with these IS6110-like nucleotide sequences (Figure 2).

The protocol for direct detection of MTC from fresh animal tissues, using an optimized DNA extraction and purification procedure coupled with a semi-nested real time PCR assay described in this work, was shown to be highly accurate and much faster than bacteriological culture, reducing the time for confirmatory TB diagnosis from several weeks to few hours, thus also potentially decreasing the arrest period of the suspect animal carcass and herd. The assay is amenable to future automation possibilities regarding both the DNA extraction and amplification steps. It may also allow the detection of MTC members when these pathogens become nonviable and non-cultivable or are overgrown by other less fastidious bacteria or mycobacteria also present in tissue samples. The use of a novel β-actin gene targeted probe, and respective flanking primers, with complementary targets in most mammal species, allowed to assess the presence of amplification inhibitors in the DNA extracts. Although the test is not able to distinguish between different members of the MTC, particularly M. bovis, the identification of any tuberculous mycobacteria infection in domestic or wildlife animals could have public health implications.

Acknowledgments

Celeste Matos, Lurdes Clemente, Madalena Monteiro, Paula Mendonça and Paulo Carvalho (INIAV, I.P.) are acknowledged for excellent technical assistance, providing bacterial strains and performing pathological analysis.