Protection conferred by homologous and heterologous whole killed and live vaccines against Brucella canis in Balb/c mice

Abstract

Investigation on vaccines against canine brucellosis is a field poorly explored. Carmichael´s pioneering work in the 1980s, evaluated a less mucoid (M-) strain of B. canis in dogs on the view of possible immunization [1]. Although the results demonstrated that the M- variant met some of the criteria for an immunizing agent, the study failed to provide unequivocal assurance of acceptable attenuation and later communications demonstrated the zoonotic nature of the strain. Furthermore, at the moment there is no available vaccine against this disease. Consequently, there are no established references for new research. Bearing in mind future evaluation of different strategies of immunization against B. canis in mouse model, we study homologous (B. canis M-) and heterologous (B.ovis REO 198) bacterins and a viable strain B. ovis PA76250 as positive control vaccines. Brucella strains were grown in Brucella Agar, harvested in sterile saline, serially diluted and bacterins were inactivated at 60°C for 60 min [2]. Forty female BALB/c mice 5-6 weeks old were distributed at random in groups of 5 mice each and were immunized with: a) 1.5x109 heat-killed B. canis M- b) 1 x109 heat-killed B. ovis REO198 and 8.5x108 live B. ovis PA76250 bacteria. Control mice were injected with saline alone. Bacterins were emulsified in Marcol 52, in Montanide IMS 3012 VG PR or in Quil A. Bacterins were injected two times with 30 days interval, by subcutaneous route. Live B. ovis were administered once subcutaneously on day 0. Thirty days after the last immunization, mice were challenged intraperitoneally with 7x106 B. canis RM6/66. Mice were sacrificed 30 days after challenged and their spleens were removed, homogenized and plated to determine colony forming units (CFU) per spleen. Results were represented as the mean log10 CFU ± SD per group. Units of protection were calculated as the difference between the mean log10 CFU from the saline group and the mean log10 CFU from the experimental groups. Live B. ovis PA76250 conferred heterologue protection (2.08 log) against B. canis. Mice receiving B. canis M- bacterin formulated in Marcol 52 or in Montanide showed similar results against virulent strain (1.19 log and 1.31 log, respectively). In conclusion, homologous bacterin or live B. ovis are interesting alternatives to be used as positive control vaccines against B. canis in future protection experiments in mouse model.