This study was part of a larger project focused on developing rapid and accurate diagnostic tools in the current framework of bTB surveillance and control programs. LN tissue samples (N=100) were collected each from a different cattle carcass at the slaughterhouse from 2018 to 2019 in the context of Spanish bTB eradication program. All samples were collected during routine post-mortem veterinary examination within an official context and according with national and European regulations. No purpose killing of animals was performed for this study, so no ethical or farmer’s consent approval was required.

LNs were independently sliced to confirm the presence of visible TBLs (N=19) or no visible lesions (NVLs) (N=81) and fixed in 10% neutral-buffered formalin for the histopathological analysis. Each LN was then individually homogenized using a tissue homogenizer (Fisherbrand, Fisher Scientific, Madrid, Spain) to obtain a uniform mixture. Tissue homogenate was divided into paired samples that were used for DNA isolation and selective microbiological culture.

For histopathological evaluation, formalin fixed LNs were processed and embedded in paraffin following standard procedures. Four-micron sections were stained with haematoxylin-eosin (H&E) and Ziehl-Neelsen (ZN). Histopathological findings were classified as TBLs for those samples with a tuberculous granuloma, pyogranuloma, or scattered Langhans-type multinucleated giant cells (MNGCs), or as no histopathological lesion (NHLs), for the tissue with normal histological characteristics and no lesion compatible with TBL (Larenas-Muñoz et al., 2022). In addition, Ziehl-Neelsen (ZN) technique was performed to detect acid-fast bacilli (AFB). A sample was considered positive for ZN when one or more AFB were found in at least one high-power field magnification (HPF, 100x). The lesions were classified as paucibacillary if it was observed with 1 to 10 AFB bacilli, or pluribacillary if ≥ 11 AFB were observed per HPF (Larenas-Muñoz et al., 2022).

For the reference standard (microbiological culture followed by real-time PCR-IS6110), tissue homogenates were decontaminated with an equal volume of 0.75% (w/v: 1/1) hexadecyl pyridinium chloride solution in agitation for 30 min. Samples were centrifuged for 30 min at 1,500 × g. The pellets were collected with swabs and cultured in liquid media (MGIT™ 960, Becton Dickinson, Madrid, Spain) using an automatized BD Bacter™ MGIT™ System (Becton Dickinson) (Corner and Trajstman, 1988). DNA extraction was performed using the MagMAX Total Nucleic Acid Isolation Kit according to the manufacturer’s instructions (Thermo Fisher Scientific, Lissieu, France). DNA was eluted in 50 μl. Then, cultures were considered positive when isolates were confirmed as MTC using real-time PCR (Thierry et al., 1990). The cut-off value of real-time PCR-IS6110 assay was set at 10 to 100 genomic equivalents, and the cut-off set at Cq ≤ 38 (10 to 100 genomic equivalents/15 μL reaction mixture) (Sánchez-Carvajal et al., 2021).

Genomic DNA isolation was conducted from tissue homogenate according to Vera-Salmoral et al., 2023 with several modifications using NucleoSpin Tissue Kit^® (Macherey-Nagel, Düren, Germany). In brief, 1 mL of homogenized tissue was centrifuged during 5 min at 9,000 g. The supernatant was discarded, and the resulting tissue pellet was added in a tube together with 250 µl of Sample Buffer T1, 150 mg of 0.5-mm glass beads and 50 mg of 0.1-mm glass beads. Then, samples were subjected to mechanical disruption (SI™ Disruptor Genie™, Scientific Industries, New York, USA) (2,850 rpm/50 Hz/20 min). After an overnight enzymatic digestion at 56°C with 30 µl proteinase K in a thermo-shaker (600 rpm/12 h), a new mechanical disruption step was conducted. Samples were centrifuged 2 min at 9,000 g, and pellets were again subjected to the steps described above. Then, samples were mixed with 200 µl of buffer T3, incubating the mixture for 10 min at 70°C. The lysate was transferred to a silica-based nucleic acid purification column and managed according to manufacturer’s instructions. Isolated DNA samples were stored at −20°C until used in downstream PCR assays. Positive and negative extraction controls were included.

Specific primers and probe were based on the homology region of the partial insertion sequence 6110 (IS6110), a repetitive mobile element specific for all the pathogens belonging to MTC widely used to diagnose and genotype this pathogen. The fluorogenic IS6110-probe was labelled with a fluorescent reporter dye [6-carboxyfluorescein (FAM)] at the 5′-end and a 3′-Black Hole Quencher 1 (BHQ1). Primers and probe used for real-time PCR and ddPCR are listed in Table 1 (Sánchez-Carvajal et al., 2021; Lorente-Leal et al., 2021).

QuantiFast^® Pathogen PCR + IC Kit (QIAGEN, Hilden, Germany) was used to conduct the real-time PCR-IS6110 evaluating each sample in duplicate in the MyiQ™2 Two-Color real-time PCR Detection System (Bio-Rad, Hercules, Ca, USA) under the following cycling conditions: 95°C for 5 min to activate the DNA polymerase followed by 42 amplification cycles that consisted of a denaturation step at 95°C for 15 s, an annealing-extension step at 60°C for 30 s. Following manufacturer’s guidelines, an exogenous inhibition heterologous control (internal amplification control, IAC) supplied with the kit was included. An inter-run calibrator with a known quantification cycle (Cq) value of 32 was introduced in each assay to self-control intra-assay. Complete inhibition of amplification was considered when IAC did not amplify and partial inhibition when it showed a Cq > 33. The analytical sensitivity or limit of detection (LOD) was estimated for the proposed primers and probes. LOD is defined as the lowest concentration in which 95% of replicates were positive, according to the Clinical and Laboratory Standard Institute guidelines. A serial 10-fold dilution series of M. bovis genomic DNA with known quantities ranging from 10⁶ to 10⁰ were used. The reactions were performed in triplicate for each dilution in three different assays. Thus, the LOD was determined to be ranging from 10 to 100 genomic equivalents, and the cut-off was established at Cq < 38 (Sánchez-Carvajal et al., 2021).

For ddPCR assay targeting IS6110, QX200™ ddPCR™ Supermix for probe (No dUTP) (Bio-Rad, Hercules, CA, USA) was used according to Bio-Rad ddPCR system guidelines. Each sample was evaluated in duplicate in a reaction mix with a final volume of 21 μl as follows: 10.5 μl of 1x ddPCR Supermix for probe (No dUTP), 1.7 μl of IS6110-forward (900 nM), 0.85 μl of IS6110-reverse (600 nM), 0.65 μl of IS6110-probe (FAM-labelled, 200 mM), 3 μl of template, and 4.3 μl of nuclease-free water. It is important to mention that several protocols for ddPCR recommend performing a restriction digestion of DNA samples outside the amplicon in order to make the template more accessible reducing sample viscosity. Nevertheless, we decided to not use restriction enzymes due to the extraction protocol herein reported got an efficient reduction of host DNA ranging from 50 – 100 ng/μl. Afterwards, the droplets were generated on the QX200 Droplet Generator (Bio-Rad, USA) using 70 μl of droplet generation oil for Probes^® (Bio-Rad, USA) dispatched into the bottom of the oil wells of the DG8™ Cartridge droplet generator (Bio-rad, USA) according to the manufacturer’s instructions. The droplets were carefully transferred to a specific 96-well ddPCR reaction plate (Bio-Rad, USA) using a RAININ Pipet-Lite Multi Pipette (Mettler Toledo, Columbus, Ohio, USA). After heat sealing by PX1™ PCR Plate Sealer (Bio-Rad, USA) at 180°C for 5 s, amplifications were run in the C1000 Touch thermal cycler (Bio-Rad, Hercules, CA, USA) under the following cycling conditions: 95°C for 10 min, followed by 40 cycles of 94°C for 30 s and 60°C (annealing/extension) for 1 min, and finally 98°C for 10 min. The temperature ramping rate was set at 2°C/s. Thereafter, the droplets were stored in darkness at 4°C for 12 h.

The limit of detection (LOD) was determined to be the lowest concentration of IS6110 copies at which detection is possible (Armbruster and Pry, 2008). LOD for ddPCR-IS6110 was determined by measuring three concentrations around LOD. Reactions were run in triplicates for each concentration (M. bovis genomic DNA with known quantities ranging from 10⁴ to 10⁰), and LOD was defined as the lowest concentration in which 95% of replicates were positive according to the Clinical and Laboratory Standards Institute guidelines. The limit of blank (LOB) was defined as the highest number of IS6110 copies found in 12 blank samples (Armbruster and Pry, 2008).

The QX200 Droplet Reader (Bio-Rad, USA) was used to read and count the fluorescent positive and negative droplets. Then, the data were analyzed using the QuantaSoft™ Analysis Pro software (version 1.0.596) (Bio-Rad, USA). Data from samples with 12,000 – 16,000 droplets were used for concentration calculations. Samples with a low number of droplets (< 10,000) were excluded from the analysis. According with the results for LOB, those samples with fewer than two positive droplets were considered “MTC-negative”, in contrast, samples were considered as “MTC-positive” when more than two droplets were found (Whale et al., 2020). Thus, the cut-off value of ddPCR-IS6110 assay was set at 3 positive droplets in 20 μL reaction mixture.

The diagnostic performance of ddPCR targeting IS6110 was evaluated for the detection of MTC in microbiological culture and fresh LN tissue samples. The diagnostic accuracy was compared with microbiological culture as the refence standard, considered as an imperfect reference technique for bTB diagnosis (Corner and Trajstman, 1988; Courcoul et al., 2014; Pucken et al., 2017). The adjusted sensitivity and specificity, false positive rate, false negative rate, positive likelihood ratio (PLR), and negative likelihood ratio (NLR) were calculated using Epidat 3.1 software. The PLR and NLR were interpreted according to the criteria published by Sackett et al. (2001), where a PLR > 10 or NLR < 0.1 indicates a technique of high diagnostic value that can discriminate between healthy and diseased animals, 5 < PLR ≤ 10 or NLR = 0.1-0.2 indicates a technique involving moderate changes in probability, 2 < PLR ≤ 5 or 0.2 < NLR ≤ 0.5 indicates a technique involving small changes in probability, and PLR ≤ 2 and NLR > 0.5 indicates rarely discernible changes. Finally, agreement between microbiological culture and ddPCR from culture, microbiological culture and ddPCR from fresh tissue, and real-time PCR and ddPCR both from fresh tissue was assessed using Cohen’s kappa coefficient (κ): κ = 0 indicated no agreement; 0.01 ≤ κ ≤ 0.20, slight agreement; 0.21 ≤ κ ≤ 0.40, fair agreement; 0.41 ≤ κ ≤ 0.60, moderate agreement; 0.61 ≤ κ ≤ 0.80, substantial agreement; and, 0.81 ≤ κ ≤ 1.00, almost perfect agreement (McHugh, 2012) (WinEpi software 2.0, Faculty of Veterinary Medicine, University of Zaragoza, Spain).

In order to optimize the ddPCR-IS6110 assay, the first step was to determine an optimal annealing temperature, considered as one of the most critical parameters. Thus, we tested a range of annealing temperatures ranging from 56°C to 64°C. A total of 50 ng of MTC DNA isolated from a selective microbiological bacterial culture, 900 nM of forward and reverse primers together with 500 nM of probe were used in the assay. No restriction digestion of the DNA samples was performed. A negative template control (NTC) containing sterile water instead of DNA was included. We were able to detect the IS6110 specific region in all the assessed temperatures ( Figure 1A ), however, the annealing temperature of 60°C (E08) showed a higher amplitude between positive and negative droplets compared with other temperatures and resulted in less non-specific amplification (rain). No positive droplets were observed in NTC for any of the temperatures ( Figure 1B ). Therefore, an annealing temperature of 60°C (E08) ( Figure 1A ) was selected as ideal temperature for further experiments.

Next step was to determine the optimal primer and probe concentrations for ddPCR-IS6110 assay. Thus, five different concentrations of forward primer (100, 400, 600, 800 and 900 nM), reverse primer (100, 300, 400, 600, 800 and 900 nM) and probe (50, 100, 200, 250 and 400 nM) were tested ( Figure 1C ). Similarly, as above mentioned, a total of 50 ng of MTC DNA isolated from a selective bacterial culture were used in the assay, with no restriction digestion of DNA samples and inclusion of a NTC with sterile water instead of DNA. Figure 1C showed that the overall fluorescence amplitude of positive droplets increased with primers and probe concentrations. On the other hand, a much better amplitude between positive and negative droplets was observed when we used a concentration of 900 nM for forward, 600 nM for reverse and 200 nM for probe ( Figure 1C , D06). This set up of primers and probes were used for further experiments.

In the case of microbiological culture, we decided to use a sample concentration of 50 ng of DNA according to the concentration and volume available after DNA extraction. In the case of DNA isolated from fresh LN tissue, we were able to test the effect of sample quantity. A ddPCR assay was run using different concentrations of DNA from two different LNs that were IS6110-positive by real-time PCR with different Cq values (500 ng, 250ng, 50 ng and 10 ng) ( Figure 2A , Cq = 26; and Figure 2B , Cq = 34). As illustrated in Figure 2A , a good separation between positive and negative droplets was observed at the four DNA concentrations (500 ng, 250ng, 50 ng and 10 ng) for DNA isolated from LN tissue samples. Because Poisson statistics test required enough negative droplets to be applied and calculate DNA concentration, we decided to use 50 ng (C12) of DNA isolated from tissue samples in further ddPCR assays. Also, this concentration could be a good fit to avoid cross-contamination in the case of samples with a high concentration of bacterial DNA.

The analytical specificity of ddPCR-targeting IS6110 was tested against some of the most common microorganisms identified in TBL, such as Mycobacterium avium subsp. paratuberculosis, Mycobacterium avium subsp. avium, Trueperella pyogenes, Streptococcus suis, and Staphylococcus spp. (Cardoso-Toset et al., 2015). All these tests yielded negative results by ddPCR-IS6110, demonstrating the specificity of the primers and probe included in the study ( Figure 2C ).

No positive droplets were found in 10 out of 12 blank samples (ddH₂O instead of DNA sample), but one positive droplet per 20 μl reaction mix was detected in two of these blank samples. Accordingly, the limit of blank was set at 1 drop/20 μl reaction, and therefore, a sample was considered as “negative” when no more than two positive droplets were obtained.

To determine the LOD, 10-fold serial dilutions of M. bovis genomic DNA with known quantities ranging from 10⁴ to 10⁰ were used. The reactions were performed in triplicate for each dilution in three different assays. MTC IS6110 sequences were detected in 100% of 10¹ dilutions assayed; however, only 50% positivity was obtained at the level of 10⁰. Thus, LOD of this ddPCR targeting IS6110 was set to 10¹ copies per 20 µl reaction.

As previously mentioned, microbiological culture positive samples need to be confirmed as MTC using real-time PCR (Thierry et al., 1990). Therefore, this part of the study compared both real-time PCR and ddPCR for the confirmation of culture positive samples. DNA was isolated from selective microbiological culture from 100 samples with gross TBL (N=19) or NVLs (N=81). Forty-nine out of 100 samples were tested as MTC-positive by real-time PCR (17 out of 19 with gross TBL) whereas 51 samples yielded a negative result and were classified as MTC-negative ( Table 2 ). The Cq values for the real-time PCR targeting IS6110 ranged from 20.00 to 36.25 (average = 29.35). No partial or complete inhibition were found in DNA isolated from microbiological culture. In the case of ddPCR targeting IS6110, forty-eight samples were found to be MTC-positive (19 out of 19 with gross TBL) and 52 MTC-negative ( Table 2 ). There was an association between real time-PCR and ddPCR, with the higher number of positive droplets, which ranged from 13,402 to 4 droplets, coinciding with those samples with a lower Cq value.

Analyzing the histopathological evaluation, 34 out of 49 samples positive to the microbiological culture by real-time PCR-IS6110 were classified as TBL whereas 15 as NHL ( Table 2 ). For the ddPCR-IS6110, histopathological TBL were found in 37 out of 48 ddPCR- IS6110-positive samples whereas 11 were classified as NHL ( Table 2 ).

DNA was isolated from homogenized fresh LN tissue samples (N=100) with gross TBL (N=19) or NVL (N=81) and subjected to both real-time PCR and ddPCR. All the samples with gross TBL were found to be MTC-positive by both real-time PCR or ddPCR targeting IS6110 ( Table 3 ). For real-time PCR-IS6110, 53 out of 100 tested samples were detected as MTC-positive and 47 as negative. The Cq values ranged from 22.96 to 38.10 (average = 32.06). A partial inhibition of IAC was found in 6 out of 100 samples, with 2 out of 5 samples revealing a positive result after dilution 1:2 and re-evaluation. In the case of ddPCR, 55 samples were tested as MTC-positive and 45 as MTC-negative ( Table 3 ). The number of positive droplets ranged from 12,618 to 3 droplets, with the highest number of positive droplets corresponding to those animals with pluribacillary lesions. As described before, an association was observed between real-time PCR and ddPCR, whereby a higher number of positive droplets corresponded to samples with higher Cq values.

According to histopathological evaluation, 40 out of 53 positive samples to real-time PCR presented histopathological TBL whereas 13 did not present microscopic lesion ( Table 3 ). Regarding ddPCR, 41 out of 55 positive samples were disclosed as positive to histopathology with 14 samples presenting NHL ( Table 3 ).

In order to validate the ddPCR-IS6110 from microbiological culture and fresh LN tissue samples, these assays were compared with the reference standard assay (selective microbiological culture confirmed by real-time PCR-IS6110). Assuming that this reference assay is considered an imperfect assay for performing MTC diagnosis (Corner et al., 2012; Courcoul et al., 2014; Pucken et al., 2017) validation was carried out using EPIDAT 3.1 software. The ddPCR-IS6110 from microbiological culture detected 44 out of 49 reference standard positive samples [Se adjusted 90.76% (95% CI: 82.58-98.96%)], and 47 out of 51 reference standard negative samples resulted to be negative for ddPCR-IS6110 [Sp adjusted = 100% (95% CI: 100%)]. Thus, an adjusted FNR of 9.23% (95% CI: 1.04-17.42%) and FPR of 0% (95% CI: 0%) were estimated ( Table 4 ). The PLR value (PLR = ∞) implies a high diagnostic value for the positive results discriminating between MTC-infected and non-infected animals. In addition, the NLR value was 0.07 meaning that it is a technique of a high diagnostic value to discriminate between healthy and diseased animals ( Table 4 ). Of note, all reference standard-negative but ddPCR-positive samples were also classified as MTC positive by histopathological evaluation or real-time-IS6110 from fresh LNs tissue ( Table 5 summaries the discordant results between different diagnostic techniques).

In the case of ddPCR-IS6110 carried out from fresh LN tissue samples, 47 out of 49 samples positive to the reference standard were found to be positive for ddPCR-IS6110 [adjusted Se 94.80% (95% CI: 88.52-100%), and 42 out of 51 reference standard-negative samples were tested as ddPCR-IS6110-negative [adjusted Sp = 100% (95% CI: 100%)] ( Table 4 ). According to these results, ddPCR-IS6110 from fresh tissue presented an adjusted FNR of 5.20% (95% CI: 0-11.50%) and FPR of 0% (95 CI: 0%) ( Table 4 ). Regardless of the true prevalence, ddPCR-IS6110 had a high diagnostic utility to confirm and discard MTC infection (PLR = ∞ and NLR = 0.05) ( Table 4 ). Noteworthy, 9 reference standard-negative but ddPCR-IS6110-positive samples were found also as positive by histopathological evaluation (8 out of 9) or real-time PCR-IS6110 from fresh LN tissue (8 out of 9) ( Table 5 summaries the discordant results between different diagnostic techniques).

Finally, Cohen’s kappa coefficient (κ) showed an almost perfect agreement between the ddPCR-IS6110 from culture and the reference standard (κ = 0.82) and a substantial agreement between the ddPCR-IS6110 from fresh LN tissue samples and the reference standard (κ = 0.76).