Limitations of Using IL-17A and IFN-γ-Induced Protein 10 to Detect Bovine Tuberculosis

Bovine tuberculosis (bTB) is primarily caused by infection with Mycobacterium bovis, which belongs to the Mycobacterium tuberculosis complex. The airborne route is considered the most common for transmission of M. bovis, and more than 15% of cattle with bTB shed the Mycobacterium, which can be detect by nested PCR to amplify mycobacterial mpb70 from a nasal swab from a cow. To screen for cytokines fostering early and accurate detection of bTB, peripheral blood mononuclear cells were isolated from naturally M. bovis-infected, experimentally M. bovis 68002-infected, and uninfected cattle, then these cells were stimulated by PPD-B, CFP-10-ESAT-6 (CE), or phosphate-buffered saline (PBS) for 6 h. The levels of interferon gamma (IFN-γ), IFN-γ-induced protein 10 (IP-10), IL-6, IL-12, IL-17A, and tumor necrosis factor alpha mRNA were measured using real-time PCR. To explore the cytokines associated with different periods of M. bovis infection, cattle were divided into three groups: PCR-positive, PCR-negative, and uninfected using the tuberculin skin test, CFP-10/ESAT-6/TB10.4 protein cocktail-based skin test, IFN-γ release assay (IGRA), CFP-10/ESAT-6 (CE)-based IGRA, and nested PCR. The expression of IP-10, IL-17A, and IFN-γ proteins induced by PPD-B, CE, or PBS was detected by ELISA. The results showed that levels of PPD-B-stimulated IL-17A and IP-10 (mRNA and protein), and CE-induced IP-10 (mRNA and protein) were significantly higher in cattle naturally or experimentally infected with M. bovis than in those that were uninfected. The levels of PPD-B- or CE-induced IL-17A and IP-10 (protein) could be used to differentiate M. bovis-infected calves from uninfected ones for 6 to 30 weeks post-infection, whereas PPD-B- and CE-induced IP-10 and IL-17A mRNA expression could be used to differentiate M. bovis-infected calves from uninfected ones between 6 and 58 weeks post-infection. However, CE-induced IL-17A (protein) was not a reliable indicator of M. bovis infection in cattle that were confirmed positive for infection by nested PCR. Furthermore, the levels of PPD-B- or CE-induced IP-10 and IL-17A protein were lower than IFN-γ in M. bovis-infected cattle. Therefore, IL-17A and IP-10 protein are not suitable biomarkers for bTB. Antigen-induced IP-10 mRNA should be analyzed further for their potential to be used in the diagnosis of bTB.

. M. bovis could infect wild animals and has a relatively wider host range than M. tuberculosis (3). Humans can also be infected with M. bovis through the ingestion of raw milk products or inhalation of aerosols (4). The airborne route of infection is considered the most common for transmission of M. bovis, and more than 15% of cattle infected with M. bovis shed the Mycobacterium (5), mainly early in infection (6). Therefore, early and accurate detection of bTB is important to control transmission of M. bovis spreading to other animals (7). The traditional bTB diagnostic methods are the tuberculin skin test (TST) and interferon gamma (IFN-γ) release assay (IGRA). Both TST and IGRA are based on the detection and comparison of cell-mediated responses induced by bovine purified protein derivatives (PPD-B) and avian purified protein derivatives (PPD-A) (8). PPD-B is obtained from a culture of virulent M. bovis, but shared antigens with non-pathogenic environmental mycobacteria can reduce the specificity of the TST (9). Although PPD-A is used in IGRA to exclude environmental Mycobacterium infections, it has failed to detect some M. bovis-infected cattle in bTB-and paratuberculosis-coprevalent dairies. Therefore, TST and IGRA based on M. bovis-specific antigens such as CFP-10 and ESAT-6 were established to obtain higher specificity in the diagnosis of bTB (8)(9)(10)(11). Considering that neither TST nor IGRA can differentiate between stages in the progression of bTB, a nested PCR assay based on the amplification of a fragment of mpb70 was established and used to detect mycobacteria in milk, nasal exudates, and bronchoalveolar lavage (BAL) fluid (12).
To develop more effective and accurate methods to diagnose tuberculosis, new candidate biomarkers have emerged recently for the diagnosis of this disease in humans and cattle. These potential biomarkers include tumor necrosis factor alpha (TNFα), interleukin-2, IL-1β, IL-17A, and IFN-γ-induced protein 10 (IP-10). IP-10 and IL-17A, particularly, have the potential to be used to differentiate between active and latent TB in humans. However, there are only a few studies on screening for bTBrelated cytokines.
In this study, we screened for cytokines related to bTB and explored whether cytokines could be used to detect M. bovisinfected cattle [including those that were PCR-positive (PCR-P) and those that were PCR-negative (PCR-N)]. First, we isolated peripheral blood mononuclear cells (PBMCs) from naturally M. bovis-infected, from experimentally M. bovis 68002-infected and from uninfected cattle, and then we stimulated the PBMCs using PPD-B, CFP-10-ESAT-6 (CE), or phosphate-buffered saline (PBS) for 6 h. The levels of IFN-γ, IP-10, IL-6, IL-12, IL-17A, and TNF-α mRNA transcripts were determined using real-time PCR. To explore whether IP-10 and IL-17A can be used to detect all M. bovis-infected cattle, cattle were divided into three groups: nested PCR-P, nested PCR-N, and uninfected cattle as determined by TST, CFP-10/ESAT-6/TB10.4 protein cocktail-based skin test, IGRA, CE-based IGRA, and nested PCR. The expression of IP-10, IL-17A, and IFN-γ proteins induced by PPD-B or CE was detected by ELISA.

MaTerials anD MeThODs ethics approval
The six Luxi beef calves used in the present study were treated carefully and according to the protocol approved by the Animal Care and Use Committee of the China Institute of Veterinary Drug Control. The animal ethics committee approval number is SYXK (2005-0021).

Bacterial species and Plasmids
Mycobacterium bovis 68002 was isolated in China and preserved in the China Institute of Veterinary Drug Control, Beijing, China. M. bovis 68002 can cause tubercles in the lungs, liver and other organs after intravenous injection into calves. It was defined as highly virulent and used to prepare bovine tuberculin in China. Bovine tuberculin (PPD-B, Harbin Pharmaceutical Group, Heilongjiang Province, China) was used in the TST. The CFP-10/ESAT-6/TB10.4 protein cocktail (0.5 mg/ml, endotoxin less than 10 EU/mg) was prepared in the Institute of Animal Sciences (IAS-CAAS), and used to make the protein cocktail-based skin test. CFP-10-ESAT-6 (20 μg/ml, CE expressed and purified in our lab, with a Trx-His-S tag at the N-terminus, with endotoxin at a concentration less than 10 EU/mg) was used in the CE-based IGRA. PET [20 μg/ml, the tag protein of the pET(32a) + vector was expressed and purified in our lab, with endotoxin at a concentration of less than 10 EU/mg] was used as control for CE. Bovine IFN-γ (500 μg/ml, expressed and purified in our lab, with a 6-His tag at the N-terminus, with endotoxin at a concentration less than 10 EU/mg) was used as a reference standard in the IGRA for the quantitative analysis of IFN-γ in bovine plasma.
Bovine iFn-γ, iP-10, and il-17a elisas All cattle tested by skin test were also tested by IGRA (Mycobacterium bovis Gamma Interferon Test Kit for cattle, Bovigam, Prionics AG, Schlieren, Switzerland) according to the manufacturer's instructions. Briefly, heparinized blood was collected from each cow and dispensed into a 24-well cell culture plate (1.5 ml/well, five wells for each cow), and stimulated with 100 µl of PPD-A (Avian Tuberculin PPD, 300 µg/ml, Prionics AG, Schlieren, Switzerland), PPD-B (Bovine Tuberculin PPD, 300 µg/ ml, Prionics AG, Schlieren, Switzerland), PBS, PET, and CE, respectively. Plasma was collected from each well after incubation for 24 h at 37°C in 5% CO2. The IFN-γ in plasma was detected using a Mycobacterium bovis Gamma Interferon Test Kit. PPD-B-simulated blood plasma having an OD value more than 0.100 above that of plasma stimulated with PPD-A and PBS, and CE-stimulated blood plasma having an OD value more than 0.100 above that of plasma stimulated with PBS indicated cattle were M. bovis-infected. The cutoff value for the CE-based IGRA was obtained in our lab by ROC analysis based on 96 uninfected cattle and 258 M. bovis-infected cattle, whose results were confirmed by nested PCR. The IFN-γ in plasma could be quantified using recombinant bovine IFN-γ as the reference standard.

nested Pcr analysis
Nested PCR was conducted to confirm the M. bovis infection as reported before (11). Briefly, nasal swabs of each cow were collected, and immersed immediately into 2 ml of sterile PBS. Then, DNA was extracted and dissolved in 20 µl of Tris buffer, and a nested PCR was performed to amplify a region of the mpb70 gene of MTC bacteria as described previously.

analysis of cytokine gene expression by real-time Pcr
Peripheral blood mononuclear cells were isolated from heparinized blood using the density gradient centrifugation method with Ficoll-Hypaque Density Solution (TIAN JIN HAO YANG Biological Manufacture Co., Ltd., Tianjin). Red blood cells (RBCs) were separated from PBMCs by RBC lysis buffer (Invitrogen, USA). After that, PBMCs were washed with PBS three times and resuspended in RPMI1640 medium [with 10% (vol/vol) fetal bovine serum, 25 mM HEPES buffer, 2 mM l-glutamine, 100 U/ml of penicillin, and 0.1 mg/ml streptomycin]. The PBMCs were seeded into a 48-well cell culture plate at 4 × 10 6 cells in a total volume of 250 µl. Following isolation, PBMCs were subsequently incubated at 37°C in 5% CO2 with PPD-B, CE, PET, or PBS for 6-8 h. After incubation, the cells were immediately lysed with 750 µl of TRIzol (Invitrogen, USA), and total RNA was isolated using an RNAeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol and eluted from the column with 50 µl of DEPC water. One microliter of RNA inhibitor (RI) was added to the RNA solution to protect against RNAase. The RNA was quantified with a spectrophotometer. One microgram of RNA was pre-mixed with 0.5 µg of Oligo dT (15) (Promega, USA) and DEPC water. A total of 12 µl of the pre-mixture was warmed in a water bath at 70°C for 5 min, and then immediately cooled in an ice bath for 5 min. The full-length cDNA was reversely transcribed in a 25-µl reaction mixture containing a pre-mixture of 5 µl of 5 × MLV Buffer (TaKaRa, Japan), 1.25 µl of 10 mM dNTP (TaKaRa, Japan), 0.5 µl of RI (TaKaRa, JPN), 1 µl of M-MLV (Promega, USA), and 5.25 µl of DEPC water. The mixture was incubated at 30°C for 10 min, followed by 42°C for 60 min, and then at 70°C for 5 min. The cDNA was stored at −80°C.
Real-time PCR was carried out with TaqMan ® Real-Time PCR Master Mix (ABI). The primer and probe sequences ( Table 1) were designed and synthesized by Shanghai GengCore Biotechnologies Co., Ltd. All reactions were performed in a 25-µl volume containing 10 µl of TaqMan Real-Time PCR Master Mix, 0.2 µl of Primer F (10 µM), 0.2 µl of Primer R (10 µM), 0.2 µl of Taqman-Probe (20 µM), and 1 µl of template cDNA. All reactions were run in triplicate and were carried out in 96-well Rxn Plate (ABI, USA) sealed with optical adhesive film (ABI, USA) on an ABI7900HT Fast Real-Time PCR System (ABI, USA). The instrument was programmed to cycle at 95°C for 2 min, followed by 40 cycles of 15 s at 95°C and then 1 min at 60°C. Relative gene expression was calculated using the 2 −ΔΔCT method, with β-actin as the reference gene, and the PBS-stimulated sample from each cow was used to calibrate PBMCs responses.
To verify the cytokines related to M. bovis infection, six male Luxi beef calves aged 1-2 months from a bTB-free dairy farm were randomly selected for testing by TST, CFP-10/ESAT-6/TB10.4 protein cocktail-based skin test, IGRA, and CE-based IGRA, and maintained in a biosafety level-3 facility. Three calves were intravenously injected with 10 6 C M. bovis 68002; another three calves were intravenously injected with PBS to serve as uninfected controls. Animals in each group were monitored for 1 year, and tested using TST and CFP-10/ESAT-6/TB10.4 protein cocktailbased skin test before injection, at 8 and 32 weeks post-infection.
These six calves were also tested using IGRA and CE-based IGRA before injection, and at 8, To explore whether PPD-B-or CE-induced IP-10, IP-17A, and IFN-γ proteins could be used to determine the stage of M. bovis infection, more than 1,000 Holstein cows were detected by TST, CFP-10/ESAT-6/TB10.4 protein cocktail-based skin test, IGRA, CE-based IGRA. The nasal swabs from M. bovis-infected cows were collected and detected by nested PCR, then the PCR-P swabs were confirmed by mycobacteria culture. The M. bovis-infected cows could be divided into two groups ( Table 2): PCR-P (21 cows determined positive for M. bovis by TST, CFP-10/ESAT-6/ TB10.4 protein cocktail-based skin test, IGRA, CE-based IGRA, and nested PCR were selected for the study) and PCR-N (21 cows determined positive for M. bovis by TST, CFP-10/ESAT-6/TB10.4 protein cocktail-based skin test, IGRA and CE-based IGRA, but negative by nested PCR were selected for the study). Heparinized whole blood from each cow was collected and stimulated with PPD-B, CE, or PBS for 24 h, and the plasma was collected from each well. The levels of IP-10, IL-17A, and IFN-γ protein expression in plasma were determined by ELISA.
The uninfected animals were from a bTB-free dairy farm and determined free from bTB by TST, CFP-10/ESAT-6/TB10.4 protein cocktail-based skin test, IGRA, CE-based IGRA, and nested PCR. All animals used in this study were determined free of paratuberculosis by Mycobacterium paratuberculosis Antibody Test Kit for Cattle (IDEXX Montpellier SAS, France) and free of brucellosis by Svanova Brucella-Ab C-ELISA (Svanova Biotech AB, Uppsala, Sweden).

Bacteriological analysis
Three M. bovis 68002-infected calves and one naturally M. bovis-infected cow were slaughtered after the experiment. Tissue samples from the lung, liver, spleen, bronchial lymph nodes, and kidney were collected and analyzed using nested PCR, Mycobacterium culture, and hematoxylin-eosin staining.

statistical analysis
Data were analyzed by analysis of variance (ANOVA) followed by Kruskal-Wallis test or Spearman correlation using GraphPad Prism 5 software (San Diego, CA, USA). A p value of <0.05 (twotailed) was considered significant.  (Figures 1A,B), whereas the levels of IL-12 mRNA were significantly higher only in PPD-B-stimulated PBMCs from M. bovisinfected cows relative to those in uninfected cows ( Figure 1A). The transcript levels of these six cytokines induced by PET were similar to those exposed to PBS, and there was no difference in these levels between M. bovis-infected and -uninfected cows ( Figure 1C). The transcript levels of these six cytokines induced by PPD-B in PBMCs from both M. bovis-infected and -uninfected cows were higher than those induced by CE (Figures 1A,B). The levels of IFN-γ, IP-10, and IL-17A transcripts induced by PPD-B or CE in PBMCs from M. bovis-infected cows were higher than those of IL-6, IL-12, and TNF-α.
Correlations among the levels of transcripts of the six cytokines were determined, and IFN-γ, IP-10, IL-17A, and TNF-α induced by PPD-B or CE showed good correlation with each other ( Table 3). IL-6 mRNA induced by PPD-B or CE showed good correlation with IL-17A, IFN-γ, and TNF-α mRNA. The levels of PPD-B-induced IL-12 significantly correlated with those of IFNγ, IP-10, IL-17A, and TNF-α, whereas the levels of CE-induced IL-12 only correlated with those of IL-6. TNF-α levels showed     (Figures 2 and 3). The mRNA transcript levels of IFN-γ, IP-10, and IL-17A induced by PPD-B or CE in PBMCs from M. bovis-infected calves were significantly higher than those from uninfected calves between 6 and 58 weeks post-infection (Figure 2). However, the levels of IFN-γ (induced by CE) and     (IFN-γ), IFN-γ-induced protein 10 (IP-10), and IL-17A (mRNA and proteins) induced by PPD-B and CE.

comparisons of cytokines between nested Pcr-P cattle and Pcr-n cattle
To assess whether IP-10 and IL-17A proteins could be used to detect all naturally M. bovis-infected cattle, more than 1,000 cows were detected by TST, CFP-10/ESAT-6/TB10.4 protein cocktailbased skin test, IGRA, and CE-based IGRA. 151 M. bovis-infected cows from dairies where bTB was prevalent and 50 uninfected cows from bTB-free dairies were selected for follow-up study. Nasal swabs from these 201 selected cows were collected and tested by nested PCR. 35 of 151 cows were determined positive by nested PCR, and 21 of these cows were confirmed positive by Mycobacteria culture and classified as PCR-P. The remaining 116 of 151 cows were determined negative by nested PCR and classified as PCR-N (data not shown). 21 PCR-P cows, 21 PCR-N cows, and 20 uninfected cows (NC) were randomly selected for additional analysis. Heparinized whole blood was collected from each cow and then stimulated by PPD-B, PPD-A, CE, or PBS for 24 h. The levels of IFN-γ, IP-10, and IL-17A protein in stimulated plasma were determined by commercial kits (Figure 4). PPD-B-induced IFN-γ, and IL-17A levels in the PCR-P and PCR-N groups were higher than those induced by CE, and significantly higher than those in the NC group. IFN-γ and IP-10 levels induced by CE in the PCR-P and PCR-N groups were significantly higher than those in the NC group. PBS-induced IP-10 levels were significantly higher in the PCR-N group than in the PCR-P and NC groups. PBS-induced IFN-γ levels were similar in the PCR-P, PCR-N, and NC groups. However, CE-and PBS-induced IL-17A levels were significantly higher in the PCR-N group than in the PCR-P and NC groups. Correlations among IFN-γ, IP-10, and IL-17A protein levels were calculated. This comparison included all individuals and all treatments (n = 62 [21 PCR-P cows + 21 PCR-N cows + 20 NC cows]). As shown in Table 5 To confirm infection, one naturally M. bovis-infected cow that was found positive by TST, CFP-10/ESAT-6/TB10.4 protein cocktail-based skin test, IGRA, CE-based IGRA, and nested PCR, was slaughtered after the experiment. There were no typical tubercles on the lung, kidney, and spleen, and hematoxylin-eosin staining of lung, kidney, spleen, and bronchial lymph node tissues indicated no pathological changes. However, the nasal swab and BAL fluid were positive by nested PCR.

ce-induced il-17a Was not a reliable indicator of Mycobacteria shedding in cattle
The airborne route of infection is considered as the most common for the transmission of M. bovis, and more than 15% of cattle with bTB shed the mycobacteria, mainly at an early stage in infection. Nested PCR based on the amplification of mpb70 can be used to detect Mycobacteria in milk, nasal exudates, and BAL fluid. Previous studies using nested PCR showed that 26% of TSTreactors shed M. bovis in nasal exudates (12). Furthermore, Flores-Villalva et al. found that in a high-prevalence herd, 60.3% (38/63) of TST-positive and 63.4% (40/63) of ESAT-6-CFP-10-based skin test-positive cattle were confirmed positive for M. bovis infection by nested PCR, whereas in a low-prevalence herd, 87.5% (7/8) of the ESAT-6-CFP-10-based skin test-positive cattle were confirmed positive by nested PCR (11). However, 24.84% (205/825) of M. bovis-infected cattle were positive by nested PCR in our previous study (9), and we found 23.18% (35/151) of M. bovis-infected cattle were positive by nested PCR in current study. The lower positivity rate in our study may be related to a difference in the bTB prevalence at diaries or the greater number of animals included in this study. There are few data on the differences in immune responses or immunopathology between cattle determined PCR positive and negative for M. bovis infection (M. bovis-infected animals diagnosed by TST or IGRA), but we did find the CE-induced and uninduced IL-17A (protein) levels were significantly higher in PCR-N cattle than in PCR-P and uninfected cattle (Figure 4). It indicated that M. bovis-infected cattle could be divided into two stages by using TST or IGRA and nested PCR results. One cow naturally infected with M. bovis in the PCR-P group was slaughtered and showed a low level of CE-induced IL-17A in plasma and no lesions in the lungs. The reason for this phenomenon is not known, but recent studies have shown that IL-17A (mRNA and protein) may be predictive of both vaccine efficacy and lesion severity when measured after vaccination and during infection, respectively (13,14). Blanco et al. also found that increased PPD-B induced IL-17A transcripts in PBMC is associated with pathology in M. bovis-infected cattle: cattle with macroscopic lesions showed a higher level of IL-17A transcripts than animals without macroscopic lesions (13). Therefore, we speculate that M. bovis-infected cattle that were PCR positive and those that were negative may be at different stages in the progression of bTB and might show different immune responses to M. bovis-specific antigens. More studies are needed to test this hypothesis.
Our study also showed that the levels of PPD-B-stimulated IL-17A (mRNA and protein) were significantly higher in cattle naturally or experimentally infected with M. bovis than in those that were uninfected. The levels of PPD-B-induced IL-17A (mRNA and protein) could be used to differentiate M. bovis-infected calves from uninfected ones for 6 to 30 weeks post-infection. However, CE-induced IL-17A (protein) was not a reliable indicator of M. bovis infection in cattle that were confirmed positive for infection

limitations of iP-10 as Biomarker of bTB
Several recent studies have shown that mycobacterial antigen (PPD-B, CFP-10-ESAT-6) -stimulated IP-10 levels are higher in patients with active TB and latent TB compared with healthy controls and to IFN-γ (15,16). Unlike IFN-γ, IP-10 levels were not age dependent, and more TB cases were identified in children aged <5 years when this chemokine was used for detection (17)(18)(19). This chemokine can even be used to detect TB in patients with HIV and those undergoing tuberculosis therapy (20)(21)(22). Therefore, IP-10 can be used as a biomarker for TB. Similarly, Goosen et al. found that in buffaloes naturally infected with M. bovis, the levels of IP-10 protein expression were significantly elevated in whole blood stimulated with ESAT-6/CFP-10 and higher than those of IFN-γ, suggesting its potential as a biomarker for bTB in African buffaloes (7,23 Previous studies have shown that levels of IP-10 induced by PPD-B or CE were significantly higher than those in IFN-γ in patients with TB and in M. tuberculosis-infected monkeys (26), whereas, in our study, levels of antigen-induced IP-10 were lower than those of IFN-γ in M. bovis-infected cattle. Because the amount of IP-10 in M. bovis-infected cattle and healthy cattle is rarely reported, the differences in results between humans, monkeys, and cattle may be related to differences in the species tested or the limited number of animals included in our study. The lower levels of PPD-B-or CE-induced IP-10 protein relative to that IFN-γ limit the utility of IP-10 for the diagnosis of bTB. The levels of PPD-B-or CE-induced IP-10 transcripts, however, should be analyzed further for their value in bTB diagnosis.
We also found that levels of PPD-B-or CE-stimulated IP-10 in PCR-P animals were similar to those in PCR-N animals, and we could not differentiate PCR-P from PCR-N animals with this chemokine. However, levels of unstimulated IP-10 (PBS exposed) in PCR-N animals were significantly higher than those in uninfected animals but similar to PCR-P animals. Similarly, studies on human TB have shown that antigen-stimulated IP-10 in plasma cannot be used to distinguish between patients with active and latent TB, whereas serum IP-10 concentrations were higher in patients with active TB than in those with latent TB. However, IP-10 is likely to be a non-specific marker of inflammation and also elevated in other bacterial, viral, and parasitic infections, whereas unstimulated serum IP-10 levels primarily reflect a proinflammatory state, therefore, unstimulated IP-10 may be limited in its use for detection of PCR-N cattle.
One drawback of this study is that we did not test every cow naturally or experimentally infected with M. bovis by nested PCR. Thus, we could not evaluate the mRNA transcript levels of cytokines in PCR-P and PCR-N animals. Another limitation is that it was impossible to slaughter all cattle used in this study to confirm the stages of bTB progression in the M. bovis-infected cattle (either PCR positive or negative). Therefore, more work is needed to determine the differences in immune responses and immunopathology between PCR-P and PCR-N cattle.