Article Figures & Data
Figures
- FIG. 1.
Identification of A. marginale strain St. Maries proteins recognized by IgG2 induced by immunization with the A. marginale subsp. centrale vaccine strain. Gels were either stained with Sypro ruby for visualization of protein (top) or used for immunoblotting to detect bound IgG2 present in serum from A. marginale subsp. centrale vaccinate (sera from vaccinate C6175) (middle). As a homologous strain positive control, immunoblotting was performed using serum from an animal infected with the St. Maries strain, vaccinate C6170 (bottom). The pH gradient for all three images is given at the top of the figure. The molecular mass in kilodaltons (kDa) is indicated on the left-hand side of the images. Circles and numbers correspond to protein spots that were identified by LC/MS and are listed in Table 1.
- FIG. 2.
Locus positions of the immunoreactive A. marginale strain St. Maries (St. M) outer membrane proteins compared with orthologues present in the sensu stricto Florida strain (FL) and Israel vaccine strain (IS) of A. marginale subsp. centrale. The complete circular chromosome is shown as linear for illustration purposes, starting with the origin of replication. AM779, AMF575, and ACIS557 are orthologues; AM854, AMF640, and ACIS486 are orthologues. msp2 ES and msp3 ES represent the positions of the msp2 and msp3 expression sites, respectively.
- FIG. 3.
Mapping of IgG2 binding to conserved membrane regions versus variant surface domains of Msp2. Ten picomoles of recombinant full-length Msp2, 9H1 hypervariable region, N-terminal conserved region, and C-terminal conserved region were immunoblotted and probed for serum IgG2 binding from each of the A. marginale subsp. centrale vaccinates: C6171 (A), C6175 (B), C6187 (C), and C6188 (D). Serum from a St. Maries strain-infected animal, C6170 (E), was used as a positive control; preinfection serum from the same animal was used as a negative control (F). Probing with anti-His antibody was used as a positive control for the presence of each recombinant protein domain (G). The position and size of molecular mass standards are indicated to the left of the images.
Tables
- TABLE 1.
Anaplasma marginale St. Maries strain proteins bound by serum IgG2 induced by live vaccination
Protein type Spot no.a Protein annotation Mascot score(s) (standard)b No. of peptides with P values of <0.05 % sequence coverage Mol wt (kDa) Theoretical pI Membrane 727 AM1220 Omp7 84 3 9 41.9 8.9 727c AM1221 Omp8 42 1 2 42.9 9.2 122, 727c AM1222 Omp9 79, 42 2, 1 6, 2 43.1 8.2 424, 425, 426 AM1255 Omp11 534, 267, 365 10, 5, 9 35, 23, 33 34.8 6.7 421, 715 AM1258 Omp13 139, 65 3, 1 6, 12 38.0 7.6 727 AM075 Omp14 30 1 2 43.1 7.6 710, 411, 712 AM779 surface-expressed protein 128, 38, 65 3, 1, 1 8, 2, 3 57.4 7.2 724 AM854 peptidoglycan-associated lipoprotein 138 3 14 25.9 5.3 123-130 AM1144 Msp2 132-640 2-16 7-16 42.1 6.0 30, 112, 113 AM1063 Msp3 593, 84, 48 14, 2, 1 14, 2, 2 104.5 6.0 Housekeeping 122 AM231 succinyl-CoA synthetase beta chain (SucC) 92 1 11 41.8 5.5 121, 122 AM254 elongation factor Tu (TufB) 647, 422 14, 8 36, 30 43.0 5.4 702 AM1131 isocitrate dehydrogenase (Icd) 40 1 11 53.5 5.8 116, 117 AM944 Hsp chaperonin (GroEL) 480, 218 9, 5 25, 18 58.4 5.2 701, 703, 411 AM1174 F1-ATP synthase alpha subunit (AtpA) 290, 188, 29 7, 3, 1 21, 7, 1 54.6 6.0 - TABLE 2.
Comparison of Anaplasma marginale subsp. centrale vaccine strain antigens with A. marginale sensu stricto orthologs
Israel vaccine strain protein annotation St. Maries strain protein annotation % identitya St. Maries strain Florida strain Mississippi strain Puerto Rico strain Virginia strain Membrane proteins ACIS_00178 Omp7 AM1220 Omp7 70, 100 68, 86 69, 94 67, 94 68, 99 ACIS_00178 Omp7 AM1221 Omp8 66, 100 64, 90 66, 90 76, 100 64, 88 ACIS_00178 Omp7 AM1222 Omp9 64, 100 64, 99 - - 64, 98 ACIS_00140 Omp11 AM1255 Omp11 60, 100 60, 97 54, 87 61, 97 - ACIS_00138 Omp13 AM1258 Omp13 63, 100 57, 98 60, 94 59, 97 - ACIS_00068 Omp14 AM075 Omp14 71, 100 71, 97 73, 99 - 71, 97 ACIS_00557 undefined product AM779 surface-expressed protein 84, 100 84, 100 84, 99 84, 100 84, 100 ACIS_00486 peptidoglycan-associated lipoprotein AM854 peptidoglycan-associated lipoprotein 79, 100 79, 100 79, 100 79, 100 79, 100 ACIS_00229 Msp2 N-terminal conserved region AM1144 Msp2 N-terminal conserved region 84, 100 84, 99 83, 99 84, 100 82, 98 ACIS_00229 Msp2 C-terminal conserved region AM1144 Msp2 C-terminal conserved region 90, 100 91, 100 86, 97 93, 97 93, 97 ACIS_00229 Msp2 hypervariable regions AM1144 Msp2 hypervariable regions 33-74b, 100 36-67, 35-100b - - - ACIS_00617 Msp3 N-terminal conserved region AM1063 Msp3 N-terminal conserved region 52, 100 51, 74 55, 77 49, 77 53,77 ACIS_00617 Msp3 C-terminal conserved region AM1063 Msp3 C-terminal conserved region 84, 100 85, 98 88, 99 78, 96 71, 88 ACIS_00617 Msp3 hypervariable regions AM1063 Msp3 hypervariable regions 18-48b, 100 13-44, 14-100b - - - Housekeeping proteins ACIS_01060 succinyl-CoA synthetase (SucC) AM231 succinyl-CoA synthetase (SucC) 98, 100 99, 100 97, 99 98, 99 - ACIS_01040 elongation factor Tu (TufB) AM254 elongation factor Tu (TufB) 97, 100 97, 100 97, 100 98, 100 97, 100 ACIS_00241 isocitrate dehydrogenase (Icd) AM1131 isocitrate dehydrogenase (Icd) 99, 100 100, 99 99, 99 100, 99 100, 99 ACIS_00394 chaperonin GroEL AM944 60-kD chaperonin (GroEL) 99, 100 99, 100 99, 100 99, 100 99, 100 ACIS_00219 Atp synthase subunit alpha (AtpA) AM1174 F1-ATP synthase alpha subunit (AtpA) 93, 100 93, 100 93, 100 93, 99 93, 100 ↵a The St. Maries, Florida, Mississippi, Puerto Rico, and Virginia strains were compared to the Israel and St. Maries strains. The first number indicates sequence identity to the Israel strain, and the second number indicates sequence identity to the St. Maries strain. The Mississippi, Puerto Rico, and Virginia strain comparisons were performed using pyrosequencing data that comprise approximately 96% of the full sequence. Only partial sequences of ortholog gene products were available (12). A dash (-) indicates a partial sequence that does not allow comparison, not the absence of the gene.
↵b For Msp2 and Msp3 hypervariable regions, a range of sequence identities is given that represents the lowest and highest sequence identities observed when comparing all donor alleles in the genome.
- TABLE 3.
Amino acid sequence identities between Anaplasma marginale subsp. centrale (Israel vaccine strain) and A. marginale strain St. Maries Msp2 hypervariable region variants
Israel vaccine Msp2 HVR variant % identity with St. Maries Msp2 HVR variant: Ψ1 Ψ2a 9H1 P1b E6/F7 G1c 36 48 58 48 63 C 38 54 49 53 54 F 37 48 51 50 42 B1 66 48 33 53 35 A22 43 40 38 38 42 AF 61 54 53 43 67 A1 74 37 43 52 46 ↵a St. Maries strain donor allele Ψ2 is duplicated, with the duplicate, 3H1, having an identical sequence (7).
↵b St. Maries donor allele Ψ P1 is duplicated, with the duplicate, G11, having an identical sequence (7).
↵c A. marginale subsp. centrale (Israel) donor allele Ψ G1 is duplicated, with the duplicate, G2, having an identical sequence (21).
- TABLE 4.
Amino acid sequence identities between Anaplasma marginale subsp. centrale (Israel vaccine strain) and A. marginale strain St. Maries Msp3 hypervariable region variants
Israel vaccine Msp3 HVR variant % identity with St. Maries Msp3 HVR variant: 1 2 3 4 5 6 7 1 30 36 42 43 33 19 29 2 22 34 53 41 42 24 40 3 26 38 31 45 39 34 41 4 30 31 19 35 34 31 34 5 21 34 48 29 32 23 34 6 24 20 27 23 32 33 23 7 27 26 31 47 44 31 33 8 22 25 37 18 30 18 30
