J Bacteriol Virol.  2015 Jun;45(2):112-125. 10.4167/jbv.2015.45.2.112.

Seroreactive Mycobacterial Proteins and Lipid in Cattle with Bovine Tuberculosis

• Jeon HS
• Shin AR
• Son YJ
• Kim JM
• Jang Y
• Kim S
• Lee KI
• Choi CH
• Park JK
• Kim HJ

• KMID: 2149211
• DOI: http://doi.org/10.4167/jbv.2015.45.2.112

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.

Keyword

Bovine tuberculosis; Antibody response; Recombinant protein; Lipoarabinomannan

MeSH Terms

Animals
Antibody Formation
Cattle*
Diagnosis
Escherichia coli
Humans
Immunoglobulin A
Immunoglobulin G
Mycobacterium bovis
Serologic Tests
Tuberculosis
Tuberculosis, Bovine*
Immunoglobulin A
Immunoglobulin G

Figure

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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.

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  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.

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  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.

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  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.

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  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.

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