Article Figures & Data
Figures
- FIG. 1.
Genomic map of PE, PPE, and UPF genes in M. avium subsp. paratuberculosis K-10 (MAP). Genes encoding PE (shown as bars inside of the circular chromosome), PPE (bars crossing the circular chromosome), and UPF (bars outside the circle) protein family members are shown. No PGRS protein-coding sequences were found. (Inset) MAP1152-MAP1156 genomic region, indicating genes encoding PPE proteins MAP1152, MAP1153, and MAP1155 (black boxes), UPF protein MAP1156 (gray box), hypothetical protein MAP1154 (dark patterned box), and MAP1150c and MAP1151c (light patterned boxes). Arrowed boxes indicate the direction of transcription.
- FIG. 2.
SDS-PAGE and immunoblot analysis of recombinant M. avium subsp. paratuberculosis MAP1152 and MAP1156 proteins. Shown is a 12% SDS-PAGE gel, stained with GelCode blue, along with three corresponding immunoblots containing purified recombinant fusion proteins. Antibody or serum samples used to probe the immunoblot are indicated beneath the panels: MBP MAb, monoclonal antibody against the maltose-binding protein; mouse 160, serum derived from a mouse immunized with live M. avium subsp. paratuberculosis cells; rabbit 273, serum derived from a rabbit immunized with live M. avium subsp. paratuberculosis cells. Size standards, reported in kDa, are indicated to the left. Assignments for the gel and blots were as follows: lane 1, protein size standards; lane 2, MAP1152; lane 3, MAP1156; lane 4, LacZ.
- FIG. 3.
Western blot analysis of antibody responses to MAP1152 and MAP1156 in naturally infected cattle. Immunoblots containing MAP1152 and MAP1156 were probed with sera from five cows (183, 2075, 84, 805, and 45) naturally infected with M. avium subsp. paratuberculosis (A) or from two additional cows experimentally infected with M. avium or M. bovis (B). Size standards, reported in kDa, are indicated to the left. Assignments for the blots were as follows: lane 1, protein size standards; lane 2, MAP1152; lane 3, MAP1156; lane 4, LacZ.
- FIG. 4.
Western blot analysis of antibody responses to MAP1152 and MAP1156 during the course of JD. Immunoblots of MAP1152 (upper gel), MAP1156 (middle gel), and K-10 whole-cell extract (lower gel) were probed with serum samples withdrawn from a naturally infected cow (cow 47) at various times during the course of the experiment: lane 1, first bleed, subclinical infection; lane 2, 12 months after first bleed, borderline clinical/subclinical infection; lane 3, 26 months after first bleed, clinical infection; lane 4, 35 months after first bleed, advanced clinical infection; lane 5, anti-MBP monoclonal antibody control. Size standards (in kDa) are indicated to the left.
- FIG. 5.
Seroreactivities of MAP1152 and MAP1156 recombinant proteins to infected and noninfected cattle. Antigen (0.010 mg) reactivity was evaluated by ELISA against sera from cattle naturally infected with M. avium subsp. paratuberculosis (filled bars) or sera from culture-negative cattle (open bars). Each serum sample was diluted 1:25 in Idexx dilution buffer. Each column represents mean absorbance (from triplicate measurements against LacZ; averaged from measurements on three different days in duplicate or triplicate for other antigens [see Table S2 of the supplemental material]) per antigen, ± standard errors of the means from noninfected cattle (left columns) or infected cattle (right columns), as evidenced from the original classification of serum samples. Significance levels are indicated as follows: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
- FIG. 6.
Individual responses of serum samples from infected and noninfected cattle to recombinant antigens. Antigen (0.010 mg) reactivity was evaluated by ELISA against sera from cattle naturally infected with M. avium subsp. paratuberculosis and sera from culture-negative cattle. Each serum was diluted 1:25 in Idexx dilution buffer. Each column represents mean absorbance (triplicate measurements against LacZ; average measurements on three different days in duplicate or triplicate for other antigens, as indicated in Table S2 of the supplemental material) per antigen, ± standard errors of the means from noninfected (−) or infected (+) cattle, as evidenced from the original classification of serum samples. The reaction to each antigen is indicated (see inset for pattern key).
Tables
- TABLE 1.
Cattle serum samples used in the study
Serum sample Original classification (Idexx test) Type of infection Source Cow 183 + Natural (JD) NADCa Cow 2075 + Natural (JD) NADC Cow 184 + Natural (JD) NADC Cow 805 + Natural (JD) NADC Cow 45 + Natural (JD) NADC Cow 193 − Experimental, with M. avium hominissius NADC Cow 2291 − Experimental, with M. bovis strain 95-1315 NADC Cow 47 + Natural (JD) NADC Positive controlb + Natural (JD) Idexx Laboratories EDNA + Natural (JD) NADC Cow 308 + Natural (JD) NADC 2010-07 + Natural (JD) Nebraska dairyc Negative controld − None Idexx Laboratories J53-90 − None Nebraska beef herdc Cow 559 − None NADC 3438-08 − None Nebraska dairyc ↵a NADC, National Animal Disease Center, Ames, IA.
↵b Positive-control serum provided with the Idexx kit, derived from a single naturally infected Holstein cow (Idexx Technical Service).
↵c Dairy or beef cattle of the University of Nebraska Lincoln Veterinary Diagnostic Center, Lincoln, NE.
↵d Negative-control serum provided with the Idexx kit, derived from a single Holstein cow residing in a herd that tested negative for paratuberculosis by whole-herd testing over multiple years (Idexx Technical Service).
- TABLE 2.
Characterizations of ORFs MAP1152 to MAP1156
ORF Size (no. of aa, mass)a H37Rv homologb/E-valuec Domain, motifa,c Comment(s)d MAP1152 416, 40.7 Rv1808 (PPE32)/8.0 E−78 PPE, GxxSVPxxW Three MH MAP1153 454, 45.7 Rv1809 (PPE33)/1.0 E−84 PPE, GxxSVPxxW Coding sequence starts 7 bp downstream from MAP1152; three MH MAP1154 117, 11.7 Rv1810/8.0 E−19 DUF732 superfamily Hypothetical protein; no MH MAP1155 320, 32.2 Rv1807 (PPE31)/4.0 E−24 PPE, GxxSVPxxW Attenuating mutation in M. tuberculosis homolog (49); two MH MAP1156 464, 50.6 Rv1425/0.0 UPF0089 M. tuberculosis homolog encodes enzyme with low TGS activity (16); possibly one MH ↵a Data are based on National Center for Biotechnology Information (NCBI) database output. Sizes are reported as the number of amino acids (aa) and as molecular mass (in kilodaltons).
↵b Data entries based on the Pfam database (http://pfam.sanger.ac.uk/family). Most homologous M. tuberculosis proteins are not necessarily orthologs (22).
↵c E-values (determined using the Blastp suite) are formatted as described in the BLAST help manual (http://www.ncbi.nlm.nih.gov/blast/blast_help.shtml).
↵d MH, membrane helices (determined via the PredictProtein server [http://www.predictprotein.org] for protein analysis [PHDhtm output]).
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
Table S1. Density data determined using Western blotting.
PDF file, 7K. - Supplemental file 2 -
Table S2. ELISA absorbance data with descriptive statistics.
PDF file, 73K. - Supplemental file 3 -
Table S3. Mixed-model regression analysis of mean absorbance differences between seropositive and seronegative samples.
PDF file, 11K. - Supplemental file 4 -
Table S4. Serum-antigen pair seroreactivity determined using GLIMMIX.
PDF file, 96K. - Supplemental file 5 -
Table S5. Percent identity and similarity of MAP1152 and MAP1156 with closest M. avium104, M. bovis, and M. avium subsp. paratuberculosis orthologs and paralogs.
PDF file, 15K.
- Supplemental file 1 -
Table S1. Density data determined using Western blotting.
