Animals used in this study were Holstein Friesian cattle aged over 1 year from (i) four dairy farms that had had no SIDT-positive cattle for more than 5 years based on repeated annual testing (n = 100), (ii) four dairy farms that had a history of BTB, but did not have any SIDT-positive animals within the current testing in a year (remote outbreak) (n = 114), and (iii) 11 farms that had at least one SIDT-positive animal within the current testing in a year (recent outbreak) (n = 260). All dairy farms were located in Northern Gyoenggi Province, Korea. SIDT-positive cattle were used as positive controls (n = 135), while animals from BTB-free farms were used as a negative control (n = 100).

Cattle were injected with 100 µL of bovine PPD (2 mg/mL) into the caudal fold, and the results of this test were based on the skin thickness determined 48 ~ 72 h after injection. The animals were considered positive if there was an increase of 5 mm or more in skin thickness, borderline-positive if the increase in skin thickness was more than 3 mm but less than 5 mm, and negative if the skin thickened by no more than 3 mm.

Heparinized blood samples were collected from each animal and delivered to the laboratory within 8 ~ 10 h of blood collection. Whole blood cultures were performed in 96-well plates in aliquots of 200 µL/well. Each aliquot was stimulated with pokeweed mitogen (PWM; Sigma-Aldrich, UK), a mixture of recombinant ESAT-6 and CFP-10 antigens, which were expressed in Escherichia coli as previously described [4,19], and phosphate buffered saline (PBS). PWM and PBS were used as positive and negative controls, respectively. The final concentration was 10 µg/mL for the antigen cocktail (5 µg/mL each of ESAT-6 and CFP-10) and 5 µg/mL for PWM. Supernatants were harvested after incubating the plates at 37℃ in a humidified 5% CO₂ incubator for 18 ~ 24 h. IFN-γ was then determined by a sandwich enzyme-linked immunosorbent assay (ELISA). Briefly, the wells were coated overnight at 4℃ with 100 µL of 1 µg/mL anti-bovine IFN-γ antibody (AbD Serotec, UK) in 50 mM carbonate buffer (pH 9.5). After blocking the wells with 10% fetal calf serum (FBS) in PBS containing 0.05% Tween (PBS-T) (assay diluent), culture supernatants were added to the wells and the samples were incubated at 4℃ overnight. After washing the plates, 100 µL of 1 µg/mL biotin-conjugated anti-bovine IFN-γ antibody (AbD Serotec) in assay diluent were added and the samples were incubated for 60 min. After further washing, 100 µL of streptavidin-horseradish peroxidase (HRP; AbD Serotec) diluted 1 : 10,000 in assay diluent were added and incubated for 30 min. After the final wash, tetramethylbenzidine (KPL, USA) was added to the wells. The reaction was stopped after 25 min by the addition of 50 µL of 2.5N H₂SO₄, at which time the absorbance at 450 nm was read. Recombinant bovine IFN-γ (AbD Serotec) was used to generate a standard curve and IFN-γ levels were reported as picograms of protein per milliliter of supernatant. Prior to analysis, the mean absorbance value from medium control wells was subtracted from that of antigen-stimulation wells. Blood culture with antigens and the IFN-γ ELISA were both run in duplicate.

Hilar lymph nodes were homogenized and treated with 2% NaOH for 15 min, then centrifuged at 3,080 × g for 15 min. Next, the supernatant was discarded, and tissue homogenates were re-suspended in PBS. The centrifugation step was then repeated and the supernatant was discarded, after which the residues were inoculated onto slopes of Ogawa medium containing 0.05% pyruvate and incubated for 12 weeks at 37℃. For DNA extraction, lymph node homogenates were prepared using a DNeasy Blood and Tissue kit (Qiagen, Germany) according to the manufacturers' instructions.

Smart Taq Pre-Mix (Solgent, Korea) was used for polymerase chain reaction (PCR) amplification, together with DNA prepared as described above and primers specific for a 113 bp IS1081 amplicon (5'-CTGCTCTCGACGTTCATCGCCG-3' and 5'-TGGCGGTAGCCGTTGCGC-3') [18]. The PCR cycle consisted of an initial denaturation step of 95℃ for 7 min, followed by 35 cycles of 30 sec at 94℃, 60 sec at 58℃, and 30 sec at 72℃, and then a final extension step of 5 min at 72℃. The PCR products were subsequently analyzed by electrophoresis with using 1.5% agarose gels (Bioneer, Korea) in 1× Tris-acetic acid-EDTA buffer (pH 7.2). A 100-bp DNA ladder (Bioneer) was used to estimate the size of the PCR products.

Data were analyzed using GraphPad Prism 5 (GraphPad Software, USA). A receiver operating characteristic (ROC) curve was generated for the ESAT-6 and CFP-10 antigen cocktail to select the cutoff values that most effectively discriminated positive from negative samples. A student's t test was used to compare the mean IFN-γ levels, and the Mann-Whitney U test was used to compare nonparametric unpaired data. A p value < 0.05 was considered to be significant.

A whole blood assay was established in a 96-well culture plate format to determine IFN-γ production after stimulation with the recombinant ESAT-6 and CFP-10 antigen cocktail. The IFN-γ concentration in each well was determined by comparing the OD values to those of wells containing recombinant IFN-γ. Standard curves were derived from 8 concentrations ranging from 0.078 ng/mL to 10 ng/mL (Fig. 1), and each plate included its own standard curve. As the samples were diluted 1 : 2 before the assay, the maximum detectable concentration was 20 ng/mL. The IFN-γ concentrations of SIDT-positive cattle were then compared with those of SIDT-negative cattle. The majority of the 135 SIDT-positive cattle produced more than 0.5 ng/mL of IFN-γ, while none of the 100 SIDT-negative cattle from BTB-free herds produced this level of IFN-γ (Fig. 2). To account for the influence of other intrinsic factors that could affect the IFN-γ response, interpretation criteria of the results were established based on the IFN-γ assay detection limit and the results in SIDT-negative control animals and the criteria of the IFN-γ assay for diagnosis of M. tuberculosis infection in humans (QuantiFERON-TB; Cellestis, Australia). In this study, animals that gave an apparently negative result and produced less than 0.16 ng/mL IFN-γ in response to the PWM mitogen or greater than 5 ng/mL in the PBS control were considered indeterminate. These criteria resulted in two cattle with indeterminate results because of low IFN-γ production, even after stimulation with the PWM mitogen, and another that showed IFN-γ production without antigenic stimulation. Therefore, these three cattle were removed from the subsequent analyses.

A receiver operating characteristics (ROC) curve was generated for the IFN-γ assay using the ESAT-6 and CFP-10 antigen cocktail to determine the cutoff value for an IFN-γ-positive result (Fig. 3). The most appropriate cutoff was defined as the point on the ROC curve with the greatest distance from the diagonal line (sensitivity = 1 - specificity), which resulted in an appropriate cutoff IFN-γ concentration of 0.125 ng/mL. However, 0.2 ng/mL of IFN-γ was used instead to improve the specificity of the IFN-γ assay without losing much sensitivity. When this cutoff was applied, 116 (85.9%) of the 135 SIDT-positive animals gave IFN-γ assay-positive results, indicating that the assay sensitivity was 85.9%. Additionally, none of the 100 SIDT-negative cattle from the BTB-free herds gave IFN-γ-positive results, indicating that its specificity was 100%.

Only SIDT-positive animals were selectively culled during annual screening; therefore, we investigated the extent of undetected M. bovis infection in the remaining cattle using the IFN-γ assay described above. To accomplish this, we tested a total of 374 animals from two groups of herds that had undergone BTB outbreaks, one consisting of 260 SIDT-negative cattle from 11 dairy herds that suffered BTB outbreaks as determined by the most recent annual test (recent outbreak), and another composed of 114 SIDT-negative cattle from four herds with a history of BTB outbreaks, but in which all of the animals tested negative in the most recent annual test (remote outbreak). The mean IFN-γ concentration of the animals from the herds with remote BTB outbreaks was significantly higher than that of animals from the herds with recent BTB outbreak (data not shown). When the cutoff criteria set by this study were applied for the IFN-γ assay, 79 (30.4%) of the 260 SIDT-negative cattle from herds with recent BTB outbreaks and 42 (36.8%) of 114 SIDT-negative animals from herds with BTB outbreaks that occurred at least 2 months previously tested positive for IFN-γ, respectively (Table 1). These results indicate that a substantial portion of animals had an infection that was not detected by the annual SIDT screen. In addition, although there was no significant difference between groups, there appeared to be a trend towards a greater number of M. bovis infections over time. However, there was marked variation in M. bovis infection rates among dairy cattle herds, regardless of the number of SIDT-positive animals (Table 2). In herd B with a recent BTB outbreak, only one (3.7%) of 27 cattle was SIDT-positive, while 20 (74.1%) were IFN-γ-positive; thus, 19 animals with M. bovis infection were not detected by SIDT. Conversely, six (16.2%) of 37cattle in herd H with a recent BTB outbreak were SIDT-positive, while seven (18.9%) of 37 cattle were IFN-γ-positive; thus, only one additional animal was identified by the developed assay. Based on the results above, total depopulation of animals in herds that have had a BTB outbreak is more suitable as a control practice.

To confirm M. bovis infection among SIDT-negative, but IFN-γ-positive cattle, we slaughtered 14 animals and examined them for the presence of visible lesions. Additionally, we removed the hilar lymph nodes for culture tests and molecular detection of M. bovis (Fig. 4). No visible lesions were found in the internal organs (including the lung, spleen, liver, and kidney), but six cattle had granuloma lesions in their hilar lymph nodes. Additionally, M. bovis was isolated from the hilar lymph nodes of five cattle, four of which had a caseous lesion. Eleven cattle, including six with caseous lesions, were M. bovis-specific IS1081 PCR positive, confirming that the IFN-γ assay used in this study could detect M. bovis in a portion of dairy cattle that were SIDT negative (Table 3).