Vaccine against infectious bovine keratoconjunctivitis: a new approach to optimize the production of highly piliated Moraxella bovis cells.

Pili are the principal antigens and virulence factors of Moraxella bovis, the etiological agent of infectious bovine keratoconjunctivitis (IBK). Although it has been reported that the low efficacy of whole cell vaccines against IBK is mainly due to the difficulties in keeping the cellular piliation level of M. bovis during the growth of bacteria in stirred bioreactors, the problem has not yet been overcome because the mechanisms involved in the loss of piliation are still not fully clarified. In this work we found that during the culture of M. bovis in liquid media, around 15% of the cells changed from piliated to non-piliated phenotypes at the end of the growth. Nevertheless, we demonstrated that the main cause of cellular piliation loss in M. bovis growing in stirred and/or sparged bioreactors is due to shear forces, which are a function of the volumetric gassed power drawn (P(g)V(-1)). Therefore, we tested here the use of bubble column bioreactors to protect M. bovis cell-bound pili from mechanical agitation damage effects. These bioreactors operated at a superficial air velocity of 0.0065 m s(-1) yielded a cellular piliation level of 25%, in contrast to 1% obtained for stirred bioreactors. The addition of carboxymethylcellulose (CMC) at 0.10% (w v(-1)) to culture medium proved to be suitable to improve the final piliation level (65%). We demonstrated by FT-IR spectroscopy and ELISA technique, that this chemical additive has a pili protective role interacting with the cells but without affecting pili antigenic properties.