In this study we have identified a 55-kDa protein, found to correspond to the AvgC protein, as one of the most constantly and intensely immunogenic proteins in M. agalactiae
. The major epitopes of this protein have been selected and characterized. The presence of the protein was scarce in the SDS-PAGE profile, but it induced a strong and constant immune response in M. agalactiae
-infected sheep. Others have indicated this protein to be a highly immunogenic surface molecule, expressed since the beginning of infection (28
). N-terminal sequencing of the 55-kDa protein showed its correspondence with the AvgC protein, encoded by the avgC
). Flitman-Tene et al. (7
) have found that the chromosome of M. agalactiae
contains a family of multiple avg
genes with significant homologies to the corresponding region of the vsp
genes in M. bovis
). Some members of the Vsp family are known to be involved in M. bovis
cytadherence to host cells (22
). Variable membrane proteins of other Mycoplasma
species, such Vaa of Mycoplasma hominis
) and Mycoplasma synoviae
A or B (MSPA or MSPB) (17
), were also shown to be adhesive. The above analogies between M. agalactiae
and M. bovis
suggest that avg
genes may be involved in cytadherence and indicate AvgC as a good candidate for immunization.
Peptide-specific antibodies eluted from selected epitopes recognized the 55-kDa protein with both epidemic strains, PCR1 and PCR2. Interestingly, with PCR1, peptide-specific antibodies also recognized a protein in the region of 26 kDa. Based on the published sequence, the expected molecular mass of AvgC would be about 26 kDa instead of 55 kDa as determined here. SDS-PAGE was performed here under strong reducing conditions (up to 15% 2-mercaptoethanol), excluding the occurrence of S-S-bond dimers to generate the 55-kDa protein. Moreover, for PG2, peptide-specific antibodies eluted from selected epitopes strongly recognized a protein in the region of 45 kDa, the same protein recognized by sera of infected sheep in immunoblotting. This suggests that the 55-kDa protein recognized in epidemic strains and the 45-kDa protein recognized in the reference strain are the same protein. Since major lipoprotein antigens of M. bovis
undergo high-frequency phase and size variations and possess extensive reiterated coding sequences (23
), it is possible that the same phenomena may occur, although the same reiterated coding sequences have never been described for M. agalactiae
. An alternative explanation would include posttranslational modifications of the AvgC protein in M. agalactiae
. Further mass spectrometry studies could clarify the relationship between these proteins recognized by the same antibodies.
The ability of mycoplasmas to overcome host immune defenses has been attributed to the rapidly changing antigenic repertoire of surface lipoproteins (19
) and to disregulation of cytokine expression (19
). Rapid changes in the antigenic repertoire of the cell surface described for mollicutes (11
) and the possible rearrangement of the avg
genes potentially involved in M. agalactiae
antigenic variation which occurs in vivo in the natural host (7
) prompted analysis of the avgC
DNA sequence of an epidemic strain isolated in Sardinia. Although the DNA homogeneity of isolates from different Italian regions was previously demonstrated by pulsed-field gel electrophoresis, suggesting that only one strain of M. agalactiae
is present in Italy (26
), the possibility cannot be excluded that in M. agalactiae avgC
variability occurs at the DNA level by analogy with vsp
genes of M. bovis
). Six hundred fifty-nine of 717 bp of the avgC
gene have been sequenced, indicating that the epidemic strain has the same sequence and differs from the reference strain PG2 in three nucleotides, resulting in three amino acid substitutions. The same substitutions have been observed in another strain (PCR2) isolated in a different year and in a different area of Sardinia, demonstrating that the mutations were conserved in epidemic strains. Two of these amino acid substitutions were within two of the five regions predicted to be most antigenic, while no substitution occurred in the epitopes selected by the immunobinding assay.
The use of highly overlapping peptides ensures that epitopes are not missed by being cut at critical points. This may occur if abutting rather than overlapping sequences are utilized. Immunoassays performed with epitopes differing only by one amino acid substitution indicate that this immunological technique is accurate for description of epitope structure. Octapeptides were used here because they reacted strongly with sera of infected sheep. The same test performed with peptides of variable length, from 9 to 12 amino acid residues and containing the selected octapeptide, showed lower reactivity. The peptide-specific antibodies α-Pep1, α-Pep2, and α-Pep3 immunoprecipitated the AvgC protein, suggesting that these epitopes are surface exposed. This may be an important requirement for their utilization in peptide vaccine development and may also be useful for diagnostic assays based on microbial capture or microbial labeling. Experiments with the peptide-specific antibodies α-Pep1, α-Pep2 and α-Pep3 against other mycoplasma species and ovine bacteria demonstrated the specificity of identified epitopes and confirmed their possible use for diagnostic purposes. The weak reaction observed with M. bovis was expected in view of homologies between the vsp gene of M. bovis and the avg gene of M. agalactiae. However, it should not represent a problem for diagnostic purposes, since the two mycoplasma species infect different hosts. The reactivities of the three epitopes with different sera (Pep2, 100%; Pep1, 60%; and Pep3, 80%) suggest that all three could be used together for immunization, while only Pep2 might be useful for diagnostic purposes.
Immunogenic epitopes described here for M. agalactiae may provide new indications for the development of single-antigen peptide or MAP vaccines and for the improvement of diagnostic tools. The use of a MAP vaccine including more than one immunogenic protein would be particularly advisable if it could be demonstrated that the AvgC protein undergoes phase variation.