Bacterial vaccine (A) for use in an animal, comprises a bacterial infective agent (B) having a knock-out mutation in each of at least three different chromosomal genes (I), where (I) encodes an immunogenic protein (IP), which serves as a marker, in (B). IP generates antibodies (Ab) that are not essential for protective immunity of the animal. Independent claims are included for the following: (1) method for preparing (A)； (2) bacterium used in (A)； (3) live or inactivated, negative-marker vaccines that include at least one (A)； (4) diagnostic method for differentiating between infected animals； animals inoculated with (A) and/or animals both naturally infected and vaccinated； (5) immunogenic proteins (IP), synthesized from at least one (B) in a vaccine (A) that generates Ab, preferably not essential for a protective immune response； (6) nucleic acid (NA) that encodes IP, including at least one mutation for matching the specific codon usage of at least one expression host, particularly Actinobacillus pleuropneumoniae or Escherichia coli； (7) host cell containing the vector of (7)； (8) preparation of recombinant IP, encoded by NA, by growing the cells of (8)； and (9) recombinant IP prepared by method (9). ACTIVITY : Antibacterial. MECHANISM OF ACTION : Vaccine.
