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Jeon, Shin, Son, Kim, Jang, Kim, Lee, Choi, Park, and Kim: Seroreactive Mycobacterial Proteins and Lipid in Cattle with Bovine Tuberculosis
Original Article

J Bacteriol Virol 2015;45(2):112-125.

Published online: 9 February 2015

DOI: https://doi.org/10.4167/jbv.2015.45.2.112

Seroreactive Mycobacterial Proteins and Lipid in Cattle with Bovine Tuberculosis

Haet Sal Jeon1, A-Rum Shin1, Yeo-Jin Son1, Jae-Myung Kim2, Yunho Jang2, Suk Kim3, Kang-In Lee1, Chul Hee Choi1, Jeong-Kyu Park1, Hwa-Jung Kim1, ∗

1Department of Microbiology and Research Institute for Medical Sciences, College of Medicine, Chungnam National University, Daejeon, Korea

2Bacteriology Disease Division, Animal and Plant Quarantine Agency, Anyang, Gyeonggido, Korea

3Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju, Korea

∗Corresponding author: Hwa-Jung Kim. Department of Microbiology, College of Medicine, Chungnam National University, 6 Munwha-Dong, Jung-Ku, Daejeon, 301-747, Korea. Phone: +82-42-580-8242, Fax: +82-42-585-3686, e-mail: hjukim@cnu.ac.kr

∗∗ This study was financially supported by the Animal and Plant Quarantine Agency (Z-1541778-2012-13-01), Ministry of Agriculture, Food and Rural Affairs, Republic of Korea.

Received 7 April 20154 May 2015       Accepted 7 May 2015

Copyright © 2015 Journal of Bacteriology and Virology

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Bovine tuberculosis caused by Mycobacterium bovis is a major economic problem in several countries. Antibody responses are useful indicators of M. bovis infection of cattle. To overcome drawback of serological tests with low sensitivity, identification and characterization of multiple serodiagnostic antigens has been required. In this study, the antigens with strong antibody reactivity were searched using fractionation of M. bovis culture filtrate proteins and probing with sera from M. bovis-infected cattle. Twelve proteins which have not previously been described as serologic targets were identified and six proteins among them were expressed in Escherichia coli. The mycobacterial lipoarabinomannan (LAM) with strong seroreactivity in cattle was identified and purified. IgG and IgA responses against the newly identified proteins, the seroreactive proteins with strong antibody reactivity in human tuberculosis, and LAM were compared in M. bovis-infected and non-infected cattle as well as in field samples. In general, sensitivity of the tested antigens was higher in M. bovis-infected cattle than purified protein derivative (PPD) (+) field samples. Although a diverse reactivity and sensitivity according to the antigens were shown, the diagnostic utility of both IgA and IgG antibody to the antigens was similar in M. bovis-infected cattle but utility of IgG antibody was superior to that of IgA in field samples. The antigen with the highest diagnostic value was LAM in both the groups. Other antigens with considerable diagnostic utility were BCG_3488c, BCG_2330, Antigen 85, HspX, and Rv3593 when considered the sensitivity and area under the receiver characteristic curve (AUC) value. These antigens may be valuable candidates to be included in a cocktail test kit for bovine tuberculosis diagnosis.

Keywords: Bovine tuberculosis, Antibody response, Recombinant protein, Lipoarabinomannan

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Figure 1.
SDS-PAGE analysis of the fractionation of Mycobacterium bovis culture filtrate proteins (CFPs). The 0~80% or 50~80% ammonium sulfate precipitate (ASP) of M. bovis AN5 total CFPs (lane 1) was fractionated into a pass fraction, 50 mM potassium phosphate buffer (kPB) eluate, and 1 mM kPB eluate by hydrophobic interaction chromatography (HIC). Three fractions were separated into a pass fraction and 500 mM kPB eluate by hydroxylapatite (HAT) chromatography, and then if necessary, further fractionated by diethylaminoethanol (DEAE) ion exchange chromatography. Total CFPs → 0~80% ASP (lane 2), total CFPs → 0~80% ASP → HIC pass (lane 3), total CFPs → 0~80% ASP → HIC 50 mM (lane 4), total CFPs → 0~80% ASP → HIC 1 mM (lane 5), total CFPs → 0~80% ASP → HIC pass → HAT pass (lane 6), M. bovis BCG total CFPs → 50~80% ASP → HAT pass → DAEA 51~66 (lane 7). The gel was stained with Coomassie blue.
jbv-45-112f1.tif
Figure 2.
2-DE and immunoblot analysis of fractionated culture filtrate proteins (CFPs). The fractions were separated by isoelectric focusing using a 7 cm pH gradient strip (pH 4 to 7) in the first dimension, and 15% SDS-PAGE in the second dimension. M. bovis AN5 total CFPs → 0~80% ASP → HIC pass → HAT pass (A), total CFPs → 0~80% ASP → HIC pass (B), total CFPs → 0~80% ASP (C), total CFPs → 0~80% ASP → HIC 50 mM (D), total CFPs → 0~80% ASP → HIC 1 mM (E) and M. bovis BCG total CFPs →50~80% ASP → HAT pass → DAEA 51~66 (F). The gels were analyzed by Coomassie blue stain and immunoblot with M. bovis-infected sera.
jbv-45-112f2.tif
Figure 3.
SDS-PAGE analysis of purified proteins. BCG_0389 (A), BCG_1909 (B), BCG_2330 (C), BCG_2765 (D), BCG_3488c (E) and BCG_3706c (F) proteins were overexpressed in Escherichia coli, purified by Ni-NTA affinity chromatography, and analyzed by SDS-PAGE with Coomassie blue staining.
jbv-45-112f3.tif
Figure 4.
SDS-PAGE analysis of purified proteins with diagnostic utility in human tuberculosis. The purified recombinant proteins of Rv3593 (A), Rv1605 (B), MAV5183 (C) and MAV4300(D) were subjected to SDS-PAGE and stained with Coomassie blue.
jbv-45-112f4.tif
Figure 5.
Antibody reactivity and purification of lipoarabinomannan (LAM). (A) The M. bovis CFPs was analyzed with Coomassie blue (CB) staining and immunoblot using M. bovis-infected cattle sera. (B) LAM from total lipid extract of M. tuberculosis was purified by Triton-X 114 phase partitioning, separated by SDS-PAGE, and then analyzed with silver staining and immunoblot using sera of M. bovis-infected (lane 1) or M. avium-infected cattle (lane 2). LM, lipomannan.
jbv-45-112f5.tif
Table 1.
Primers sets for genes amplified by polymerase chain reaction (PCR)
Protein Primer sequences (5′ – 3′) Restriction site used
BCG_0389 5′ CATATGGCTCGTGCGGTCGGGATCGAC 3′ Nde I
5′ AAGCTTCTTGGCCTCCCGGCCGTCGTC 3′ Hind III
BCG_1909 5′ CATATGAAGTCAGCAAGCGACCCGTTC 3′ Nde I
5′ AAGCTTTGTGACCGCCAGTAATGCCAC 3′ Hind III
BCG_2330 5′ CATATGGCCACCAGTAGCGACGACATC 3′ Nde I
5′ AAGCTTTGGGCCGACAGGAAGCTCAGC 3′ Hind III
BCG_2765 5′ CATATGCCCGTCGTCGTCGTCGCCACG 3′ Nde I
5′ AAGCTTCCGGCGCAGCTGCCCTTTGCT 3′ Hind III
BCG_3488c 5′ CATATGGCGAAGGTGAACATCAAGCCA 3′ Nde I
5′ AAGCTTCTTGGAAACGACGGCCAGCAC 3′ Hind III
Rv1605 5′ GGGCCCCATATGTATGCCGACCGTGACCTTCCGGGG 3′ Nde I
5′ GGGCCCAAGCTTTCGCACGGTGATTCCTTCCGCGGC 3′ Hind III
Rv3593 5′ CGCCATATGAATCACGGGCACCGGATCGAC 3′ Nde I
5′ CCCAAGCTTTTGTGGCGCTATCAACGCAAAG 3′ Hind III
MAV5183 5′ CATATGTCCAAGCCGGGG CTTCCGGTG 3′ Nde I
5′ AAGCTTGGTGGCGGGCTGGGCGGGTTG 3′ Hind III
MAV4300 5′ GGGCCCCATATGTCTGTCGAGCCGGACGTCGAA 3′ Nde I
5′ GGGCCCCTCGAGGCGGGAGAACATGATGGCGCG 3′ Xho I
Table 2.
Identification of Mycobacterium bovis seroreactive proteins by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS)
Spot No. Protein Mass (kDa) pI Amino acid (aa) Locus tag
J1 Conserved hypothetical protein 31.567 11.92 288 BCG_3953c
J2 Possible transposase 25.021 11.39 225 BCG_3456
J3 Probable transposase (fragment) 23.975 11.41 228 BCG_1093c
J4 Hypothetical protein BCG_2330 10.598 9.35 95 BCG_2330
J5 Conserved hypothetical protein 16.207 7.79 145 BCG_1909
J6 Putative conserved lipoprotein lpqU 25.983 6.96 243 BCG_1079
J7, 8, 9 Secreted antigen 85A precursor 35.686 6.1 338 BCG_3866c
J10 Conserved hypothetical protein 69.775 4.7 677 BCG_1871c
J11 Probable chaperone protein dnaK 66.83 4.85 625 BCG_0389
J12 Probable membrane protein 23.116 6.81 209 BCG_2817
J13 Chain A, crystal structure of cpn10, GroES 10.667 4.6 100 BCG_3488c
J14 Cold shock protein A 7.336 5.2 67 BCG_3706c
J15 Hypothetical protein Rv2749 11.149 5.2 104 BCG_2765
Table 3.
IgG antibody responses to each antigen in sera from Mycobacterium bovis-infected cattle and non-infected cattlea
Antigens Groups (n=60)
M. bovis-infected cattle (n=30) Non-infected cattle (n=30)
Mean OD ± SD % sensitivity (no. of positive samples) AUC (μg · h/ml) (95% CI) P valueb Mean OD ± SD % specificity (no. of positive samples)
BCG_0389 0.0218±0.0206 23.3 (7) 0.6422 (0.5007~0.7838) 0.0585 0.0124±0.0132 90.0 (3)
BCG_2330 0.1664±0.0657 3.3 (1) 0.6289 (0.4854~0.7724) 0.0864 0.1252±0.092 100.0 (0)
BCG_2765 0.0021±0.0129 16.7 (5) 0.6778 (0.5395~0.8160) 0.018 0.0001±0.0094 96.7 (1)
BCG_3488c 0.0065±0.0102 3.3 (1) 0.7489 (0.6226~0.8752) 0.0009 0.0018±0.012 96.7 (1)
Ag85 0.0738±0.2035 53.3 (16) 0.8450 (0.7487~0.9413) <0.0001 0.0002±0.0058 96.7 (1)
HspX 0.0613±0.2137 26.7 (8) 0.7711 (0.6531~0.8891) 0.0003 0.0061±0.0082 93.3 (2)
CysA 0.0137±0.0129 40.0 (12) 0.8689 (0.7764~0.9613) <0.0001 0.0017±0.0057 96.7 (1)
Rv1605 0.0076±0.0133 30.0 (9) 0.7294 (0.5980~0.8609) 0.0023 0.0002±0.0054 96.7 (1)
Rv3593 0.0229±0.0538 33.3 (10) 0.7206 (0.5895~0.8516) 0.0034 0.003±0.0059 96.7 (1)
MAV5183 0.2726±0.2444 6.7 (2) 0.6178 (0.4736~0.7620) 0.1171 0.3199±0.1582 100.0 (0)
MAV4300 0.0365±0.0351 10.0 (3) 0.5550 (0.4072~0.7028) 0.4643 0.0306±0.0278 93.3 (2)
LAM 0.2118±0.2713 90.0 (27) 0.9600 (00.9051~1.015) <0.0001 0.0105±0.007 96.7 (1)

a The cut-off value for each antigen was calculated as mean optical density (OD) plus two standard deviations (SD) obtained with sera from cattle known to be non-infected with M. bovis. AUC = area under the receiver characteristic curve; CI = confidence interval.

b Mann-Whitney test was used to determine differences between two groups for each antigen.

Table 4.
IgA antibody responses to each antigen in sera from Mycobacterium bovis-infected cattle and non-infected cattlea
Antigens Groups (n=60)
M. bovis-infected cattle (n=30) Non-infected cattle (n=30)
Mean OD ± SD % sensitivity (no. of positive samples) AUC (μg · h/ml) (95% CI) P valueb Mean OD ± SD % specificity (no. of positive samples)
BCG_0389 0.1447±0.2052 20.0 (6) 0.6889 (0.5542~0.8235) 0.012 0.0735±0.0448 96.7 (1)
BCG_2330 0.0722±0.2118 33.3 (10) 0.7833 (0.6704~0.8963) 0.0002 0.012±0.0105 96.7 (1)
BCG_2765 0.0625±0.0686 20.0 (6) 0.6272 (0.4852~0.7692) 0.0906 0.0331±0.0344 96.7 (1)
BCG_3488c 0.0792±0.2108 50.0 (15) 0.7856 (0.6702~0.9009) 0.0001 0.0149±0.0098 100.0 (0)
Ag85 0.0808±0.2209 40.0 (12) 0.7767 (0.6578~0.8956) 0.0002 0.0075±0.0102 93.3 (2)
HspX 0.0252±0.0225 10.0 (3) 0.6672 (0.5281~0.8063) 0.0261 0.0161±0.0209 90.0 (3)
CysA 0.0083±0.0263 10.0 (3) 0.7189 (0.5841~0.8537) 0.0036 0.0001±0.0213 96.7 (1)
Rv1605 0.0973±0.2102 36.7 (11) 0.7767 (0.6610~0.8923) 0.0002 0.0233±0.0166 100.0 (0)
Rv3593 0.0507±0.041 40.0 (12) 0.7883 (0.6759~0.9007) 0.0001 0.0192±0.0137 96.7 (1)
MAV5183 0.2564±0.2707 20.0 (6) 0.5633 (0.4160~0.7107) 0.3994 0.1774±0.0881 96.7 (1)
MAV4300 0.1079±0.2081 30.0 (9) 0.7667 (0.6485~0.8849) 0.0004 0.0286±0.0349 96.7 (1)
LAM 0.2164±0.3078 60.0 (18) 0.8856 (0.8032~0.9679) <0.0001 0.0247±0.0213 93.3 (2)

a The cut-off value for each antigen was calculated as mean optical density (OD) plus two standard deviations (SD) obtained with sera from cattle known to be non-infected with M. bovis. AUC = area under the receiver characteristic curve; CI = confidence interval.

b Mann-Whitney test was used to determine differences between two groups for each antigen.

Table 5.
IgG antibody responses to each antigen in field samplesa
Antigens Group (n=155)
PPD (+) cattle (n=59) PD (-) cattle (n=96)
P Mean OD ± SD % sensitivity (no. of positive samples) AUC (μg · h/ml) (95% CI) P valueb P Mean OD ± SD % specificity (no. of positive samples)
BCG_0389 0.0253±0.0258 5.1 (3) 0.5280 (0.4277~0.6284) 0.56 0.0225±0.0224 96.9 (3)
BCG_2330 0.0806±0.0648 18.6 (11) 0.6245 (0.5316~0.7174) 0.0097 0.0514±0.0448 95.8 (4)
BCG_2765 0.0033±0.061 0.0 (0) 0.5580 (0.4444~0.6715) 0.2282 0.0021±0.0307 99.0 (1)
BCG_3488c 0.0191±0.0197 55.9 (33) 0.6956 (0.5989~0.7924) <0.0001 0.0026±0.0073 95.8 (4)
Ag85 0.0072±0.014 13.6 (8) 0.6724 0.5867~0.7581) 0.0003 0.0011±0.0088 94.8 (5)
HspX 0.049±0.2823 10.2 (6) 0.5927 (0.5039~0.6815) 0.0531 0.0099±0.0152 95.8 (4)
CysA 0.0077±0.0062 1.7 (1) 0.7071 (0.6263~0.7879) <0.0001 0.0042±0.0113 96.9 (3)
Rv1605 0.0034±0.0061 0.0 (0) 0.6326 (0.5454~0.7198) 0.0057 0.0086±0.0114 93.7 (6)
Rv3593 0.0025±0.0092 8.5 (5) 0.5535 (0.4608~0.6462) 0.2665 0.0028±0.0068 96.9 (3)
MAV5183 0.4782±0.425 33.9 (20) 0.6613 (0.5597~0.7629) 0.0008 0.2652±0.2073 94.8 (5)
MAV4300 0.0326±0.03 8.5 (5) 0.5767 (0.4849~0.6685) 0.1108 0.0262±0.0271 94.8 (5)
LAM 0.2129±0.2007 71.2 (42) 0.9328 (0.8915~0.9740) <0.0001 0.019±0.0206 96.9 (3)

a The cut-off value for each antigen was calculated as mean optical density (OD) plus two standard deviations (SD) obtained with sera from purified protein derivative (PPD) (-) cattle. AUC = area under the receiver characteristic curve; CI = confidence interval.

b Mann-Whitney test was used to determine differences between two groups for each antigen.

Table 6.
IgA antibody responses to each antigen in field samplesa
Antigens Group (n=155)
PPD (+) cattle (n=59) PPD (-) cattle (n=96)
Mean OD ± SD % sensitivity (no. of positive samples) AUC (μg · h/ml) (95% CI) P valueb Mean OD ± SD % specificity (no. of positive samples)
BCG_0389 0.0961±0.2789 13.6 (8) 0.5717 (0.4760~0.6673) 0.1347 0.0685±0.0398 93.7 (6)
BCG_2330 0.0547±0.2815 6.8 (4) 0.6705 (0.5870~0.7539) 0.0004 0.0117±0.0158 95.8 (4)
BCG_2765 0.0592±0.0393 3.4 (2) 0.5395 (0.4470~0.6319) 0.4102 0.0565±0.0458 97.9 (2)
BCG_3488c 0.0629±0.2816 3.4 (2) 0.5200 (0.4286~0.6113) 0.6771 0.0285±0.0302 91.7 (8)
Ag85 0.0562±0.2816 6.8 (4) 0.5640 (0.4733~0.6547) 0.1816 0.0175±0.0208 94.8 (5)
HspX 0.0201±0.0165 3.4 (2) 0.5200 (0.4257~0.6144) 0.6758 0.0223±0.0186 95.8 (4)
CysA 0.006±0.0325 1.7 (1) 0.5560 (0.4610~0.6509) 0.2445 0.0126±0.0392 95.8 (4)
Rv1605 0.0817±0.2792 1.7 (1) 0.5514 (0.4574~0.6453) 0.2836 0.0532±0.036 96.9 (3)
Rv3593 0.04±0.0299 5.1 (3) 0.6312 (0.5369~0.7255) 0.0062 0.0511±0.0332 95.8 (4)
MAV5183 0.12±0.1248 1.7 (1) 0.5672 (0.4735~0.6608) 0.1627 0.1755±0.229 95.8 (4)
MAV4300 0.1026±0.2809 3.4 (2) 0.5245 (0.4279~0.6210) 0.6098 0.0779±0.0695 96.9 (3)
LAM 0.2026±0.3073 49.2 (29) 0.8397 (0.7694~0.9100) <0.0001 0.0372±0.0339 97.9 (2)

a The cut-off value for each antigen was calculated as mean optical density (OD) plus two standard deviations (SD) obtained with sera from purified protein derivative (PPD) (-) cattle. AUC = area under the receiver characteristic curve; CI = confidence interval.

b Mann-Whitney test was used to determine differences between two groups for each antigen.

Table 7.
Effect of antigen combination to increase the sensitivity
Ag combination M. bovis-infected /non-infected cattle
Sensitivity % positive sera) (No. of Specificity % (No. of positive sera)
IgG    
LAM only 90.0 (27) 96.7 (1)
LAM + Ag85 93.3 (28) 93.3 (2)
IgA    
LAM only 60.0 (18) 93.3 (2)
LAM + BCG_3488c 70.0 (21) 93.3 (2)
     
Ag Combination PPD (+) and (-) field sample
Sensitivity % (No. of positive sera) Specificity % (No. of positive sera)
IgG    
LAM only 71.2 (42) 96.9 (3)
LAM + BCG_3488c 84.7 (50) 93.7 (6)
IgA    
LAM only 49.2 (29) 97.9 (2)
LAM + Ag85 50.8 (30) 94.8 (5)
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