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Five BALB/c mice were immunized by intraperitoneal injection of the 200 μl suspension containing 108 inactivated bacteria per ml or OMPs mixed with an equal volume of Freund's adjuvant (complete Freund's adjuvant for the first immunization and incomplete for the second and third immunization) for three times at 2-week intervals.

The immunogen was emulsified with complete Freund's adjuvant (Sigma) for the first immunization.

For the first immunization, 50 μg antigen and complete Freund's adjuvants (Difco, USA) were used.

The T helper1 (Th1) oriented response persisted for 12 weeks after the first immunization.

The mice were observed for 6 weeks after the first immunization.

The vaccine and dose assigned during the first immunization were maintained for second and third immunizations.

For histological analysis, mice were sacrificed at day 66 after the first immunization.

Each rabbit was bled before the first immunization and one week after each boost for the isolation of serum.

After the first immunization, the titers of anti-rOmpF against rOmpF were 1 100 1 300, and rised sharply to 1 27,000 1 240,000 after the second and third immunization (Fig. 4A).

45 days after the first immunization, rabbit serum containing antibody against β-1,3-1,4-glucanase β-1,3-1,4-glucanase β-1,3-1,4-glucanase β-1,3-1,4-glucanaser 15 min.

The antibody titers were determined after the first immunization.