1. As far as could be ascertained, this is the first report mapping the heamagglutinating activity of a M. synoviae vlhA gene. The finding that the antiserum raised against this C-terminal region inhibited the haemagglutinating activity of the homologous M. synoviae culture, definitely confirmed that the surface exposed C-terminal
60 residues of MS2/28.1 is associated with haemagglutination. It remains to be seen whether other regions of MS2/28.1 contribute to haemagglutination. The results described above highlight the extent to which vlhA genes could vary, in both the size and the sequence composition, without compromising their haemagglutination activity. Hence, comparing the expressed sequences AZ 628 datasheet from several SBI-0206965 naturally evolved haemagglutinin variant clones may help identifying critical residues involved in the haemagglutinating activity of vlhA. These variations would enable the bacterium to expose an antigenically highly divergent product to better escape the immune system
[6, 17]. Such a plausible consequence is presently under investigation. However, we anticipate that, during natural infection, in the face of the immune pressure, such an antigenic shift may occur frequently. It would thus be of interest to perform sequence comparisons between naturally derived vlhA gene sequences by focusing on their variable haemagglutinin portion. Finally, because site-specific recombination events within vlhA genes occur frequently through in vitro Calpain culture passages, inter-laboratory variations in M. synoviae stocks that had been colony purified are likely to exist. Conclusions The present study provided an indication of the extent to which the vlhA haemagglutin gene of M. synoviae could vary without compromising the surface exposure and the haemagglutinating activity of its encoded product. We thus anticipate that the antigenic repertoire of M. synoviae vlhA gene could be much wider than previously thought. Methods Bacterial strains, plasmids and culture conditions Mycoplasma synoviae strain WVU
1853 was obtained from the American Type Cell Culture collection (ATCC 25204 ) and grown in Frey’s medium [19] supplemented with 15% (v/v) foetal calf serum. The strain was initially passaged in vitro at least 7 times before being subjected to three colony purification steps. A single colony was selected and grown. All mycoplasma cultures were then prepared from this primary stock and never exceeded two additional passages. Culture conditions and antigen preparation were performed as described elsewhere [20, 21]. The mycoplasma antigens were stored at -20°C until they were needed either for Western blot, RNA or DNA extraction protocols. The growth of E. coli strains was carried out in LB or 2YT broths [22].