Article Figures & Data
Figures
- FIG 1
A 6-kbp region of the MBOVPG45_710 (milA) gene was cloned as two 3-kbp fragments, AB and CD, and subcloned as four 1.5-kbp DNA fragments, A, B, C, and D, with the expressed gene sequences bounded by restriction endonuclease cleavage sites. In addition, two 2-kbp DNA fragments were amplified using primers containing appropriate restriction endonuclease cleavage sites: abF-BamHI (CAACggatccATCAAAGACGTGA [binding site, 144–166]) and abR-SalI (TTCgtcgacGGATTTCGCCT [1931–1950]) and cdF-BamHI (CACTGggatccATCTCGAACATC [3128–3150]) and cdR-SalI (ACAgtcgacCCAGGTTCG [4955–4872]).
- FIG 2
Western blots of whole-cell proteins of M. bovis strain 3683 probed with sera from 6 calves (A through F) collected on day 0 (lanes 1) and day 24 (lanes 2). Uninfected, calves inoculated with mycoplasma culture medium; infected, calves inoculated with M. bovis strain 3683; arrow, protein of interest seen in strip 2 in infected calves.
- FIG 3
SDS-PAGE and Western blot analysis of MilA (226 kDa). Lane 1, Spectra multicolor high-range protein ladder (Fermentas); lane 2, whole-cell proteins of M. bovis strain 3683 stained with Coomassie blue; lane 3, whole-cell proteins of M. agalactiae strain PG2 stained with Coomassie blue; lane 4, Western blot of whole-cell proteins of M. bovis strain 3683 probed with pooled M. bovis-specific calf sera; lane 5, Western blot of whole-cell proteins of M. agalactiae strain PG2 probed with pooled M. bovis-specific calf sera.
- FIG 4
Physical map of genes in the region surrounding MBOVPG45_0710 (milA). The predicted size of the gene product is shown as the number of amino acids (aa). LP, lipoprotein; rpoB, DNA-directed RNA polymerase subunit beta; rpoC, DNA-directed RNA polymerase subunit beta; MBOVPG45_0710 (milA), membrane protein; HP, hypothetical protein; ISMbov 7, truncated transposase; ISMbov 6, transposase; peptidase, M42 family glutamyl aminopeptidase.
- FIG 5
Release of resorufin by the lipase activity of GST-ab over 30 min, with GST as the negative control.
- FIG 6
Log10 IgG antibody titers of individual calves on day 17 (i) and day 24 (ii) after infection in experiment 1. The bar indicates the mean of each group, and the dashed line indicates the cutoff point. *, significantly different.
- FIG 7
Log10 IgG antibody titers of individual calves on (i) day 17 and (ii) day 24 after infection in experiment 2. The bars indicate the means of each group, and the dashed lines indicate the cutoff points. *, significantly different.
- FIG 8
Log10 IgM antibody titers of individual calves on day 17 (i) and day 24 (ii) after infection in experiment 2. The bars indicate the means of each group, and the dashed lines indicate the cutoff points. *, significantly different.
Tables
- TABLE 1
Homologs of milA in other mycoplasma species
Organism Gene name Predicted gene function Query coverage (% of residues) Amino acid sequence similarity (%) M. bovis PG45 MBOVPG45_0710 (milA) Membrane protein 100 100 M. bovis Hubei-1 MMB_0654 Conserved hypothetical protein 99 92 M. agalactiae 5632 MAGa6830 Conserved hypothetical protein 100 89 M. agalactiae PG2 MAG_6100 Conserved hypothetical protein 100 89 M. bovis Hubei-1 MMB_0318 Conserved hypothetical protein 99 75 M. columbinum SF7 MCSF7_01871 Lipase 99 48 M. fermentans JER MFE_02570 Lipase 97 49 M. fermentans M64 MfeM64YM_0307 Hypothetical protein 97 49 - TABLE 2
Anti-M. bovis IgG titers of calf sera from experiment 1
Group no. Infection status No. of calves Mean ± SD of log10 anti-M. bovis IgG titers at the indicated day after challengea 10 17 24 1 Uninfected 4 1.39 ± 0.2 A 1.4 ± 0.1 A 1.35 ± 0.14 A 2 Infected 12 1.28 ± 0.2 A 1.39 ± 0.3 A 2.19 ± 0.37 B ↵a Values marked with the same letter in the same column are not significantly different (P < 0.05).
- TABLE 3
Anti-M. bovis IgG titers of calf sera from experiment 2
Group no. Infection status No. of calves Mean ± SD of log10 anti-M. bovis IgG titers at the indicated day after challengea 10 17 24 1 Uninfected 5 1.4 ± 0.38 A 1.28 ± 0.4 A 1.27 ± 0.3 A 2 Infected 30 1.15 ± 0.25 A 1.3 ± 0.4 A 2.3 ± 0.4 B ↵a Values marked with the same letter in the same column are not significantly different (P < 0.05).
- TABLE 4
Anti-M. bovis IgM titers of calf sera from experiment 2
Group no. Infection status No. of calves Mean ± SD of log10 anti-M. bovis IgG titers at the indicated day after challengea 10 17 24 1 Uninfected 5 1.9 ± 0.25 A 1.8 ± 0.1 A 1.9 ± 0.3 A 2 Infected 30 2.0 ± 0.28 A 2.3 ± 0.3 B 2.6 ± 0.3 B ↵a Values marked with the same letter in the same column are not significantly different (P < 0.05).
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
Fig. S1. Expression of recombinant proteins in E. coli JM 109 cells. Fig. S2. Multiple sequence alignment of region 1 of mycoplasma immunogenic lipase (MilA) with homologues in other mycoplasmas and the mycoplasma GDSL carboxyesterases.
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- Supplemental file 1 -
