This study evaluated the immunogenicity and dose-dependent immune response of toxinotypes of bovine origin viz Staphylococcus aureus (tst), Streptococcus uberis (Cpn-60 targeted STUB), and Escherichia coli (aggR) in a rabbit model – a step forward towards an effective polyvalent mastitis vaccine. To evaluate the primary and secondary immune response (immunogenicity) against the concentration of 10⁶ cells/mL of each preparation containing subject vaccinal isolates, 24 rabbits were divided randomly into 4 equal groups viz A, B, C and D. Each antigenic preparation was inoculated to the rabbits of group A to C @ 0.2 mL SC while group D served as control (placebo) at day 0 as priming dose and a booster dose at day 7 respectively. The serum antibodies titers were recorded in terms of Optical Density (OD) values at day 0 (pre-inoculation), 7, 14, 21 and 28 (post-inoculation) by iELISA Mastitis Kit (Abbexa, UK). The antibody response was significantly higher (p<0.05) in the post-booster samples at day-14 and day-21 for all the selected isolates. The highest primary antibody response (1.74 ± 0.14) as well as secondary immune response (2.04 ± 0.13) was shown by Strept. uberis (Cpn-60 STUB) in group B on day-14 followed in order by group C and A respectively. Likewise, dose-dependent immune response to composite antigens of 3 different antigenic concentrations of vaccinal isolates: 10⁸ cells/mL; 10¹⁰ cells/mL; 10¹² cells/mL of each of vaccinal isolates in 18 adult rabbits divided randomly into 3 groups of 6 (A, B and C) respectively for dose standardization was evaluated. Serum samples were collected at weekly intervals following completion of 2^nd shot of inocula of each dose for 3 consecutive weeks. The rabbits of group B injected with 10¹⁰ cells mL^-1 of S. aureus (tst) showed a significantly higher (p<0.05) serum iELISA O.D value (2.97^{A, a} ± 0.10) followed in order by E. coli (aggR) (2.84^a ± 0.08) and Str. uberis (cpn60 STUB) (2.78^a ± 0.07) at day-14 post-inoculation. This study revealed the concentration of 10¹⁰ cells/mL of each subject mastitogens as a standard bacterial load for polyvalent mastitis vaccines with higher and sustained antigenicity in rabbits.