Bacterial vaccine

A method for producing a bacterial vaccine composition against a bacterial disease in a tetrapod and/or a fish, wherein said bacterial vaccine composition comprises inactivated bacteria of the bacterium causing said bacterial disease, said method comprising:

• i) preparing cultures A, B, C, D, F, G and H, or preparing all of cultures A to H, wherein:
• culture A is prepared by first preparing a pre-culture “a” by inoculating said bacterium in 0.5 to 3.5% NaCl, and incubating before transferring said pre-culture “a” to a nutrient-rich bacterial growth medium with 0.5% to 3.5% NaCl, and incubating under microaerophilic conditions to prepare culture A;
• culture B is prepared by first preparing a pre-culture “a” by inoculating said bacterium in 0.5 to 3.5% NaCl and incubating before transferring said pre-culture “a” to cell culture medium for fish or mammalian cells, and incubating under microaerophilic conditions to prepare culture B;
• culture C is prepared by first preparing a pre-culture “b” by inoculating said bacterium in a cell culture medium for fish or mammalian cells, and incubating before transferring said pre-culture “b” to a solution typically comprising about 0.5 to 15% (w/v) gelatin with 0.05 to 1.5% glucose and incubating under microaerophilic conditions to prepare culture C;
• culture D is prepared by first preparing a pre-culture “c” by inoculating said bacterium in blood, plasma or serum and incubating before transferring said pre-culture “c” to brain heart infusion medium with 0.05 to 1.5% glucose and incubating under microaerophilic conditions to prepare culture D;
• culture E is prepared by inoculating said bacterium in enrichment broth and incubating under aerobic conditions to prepare culture E;
• culture F is prepared by transferring bacteria from culture E or from enrichment medium containing for example blood, plasma and/or serum to 0.5 to 3.5% NaCl;
• culture G is prepared by transferring bacteria from culture E or from enrichment medium containing for example blood, plasma and/or serum, to a cell culture medium for fish or mammalian cells;
• culture H is prepared by transferring bacteria from culture E or from enrichment medium containing for example blood, plasma and/or serum to blood or plasma serum;
• ii) inactivating the bacteria of the prepared cultures;
• iii) optionally washing the inactivated cultures of step ii);
• and
• iv) mixing the optionally washed and resuspended inactivated cultures to prepare the bacterial vaccine composition;
• wherein step iv) of mixing optionally may be performed before step ii) or before step iii) instead of after step iii).