We have constructed a defined acapsular mutant in Pasteurella multocida X-73 (serogroup A:1) by disrupting the hexA gene through the insertion of a tetracycline resistance cassette. The genotype of the hexA::tet(M) strain was confirmed by PCR and Southern hybridization, and the acapsular phenotype of this strain was confirmed by electron microscopy. The hexA::tet(M) strain was attenuated in both mice and chickens. Complementation of the mutant with an intact hexAB fragment restored lethality in mice but not in chickens. In contrast to the results described previously for P. multocida serogroup B (J. D. Boyce and B. Adler, Infect. Immun. 68:3463-3468, 2000), the hexA::tet(M) strain was sensitive to the bactericidal action of chicken serum, whereas the wildtype and complemented strains were both resistant. Following inoculation into chicken muscle, the bacterial count of the hexA::tet(M) strain decreased significantly, while the wild-type and complemented strains both grew rapidly over 4 h. The capsule is thus an essential virulence determinant in the pathogenesis of fowl cholera.Pasteurella multocida is associated with a wide range of diseases in many species of animals, the major diseases being hemorrhagic septicemia (HS) in ungulates, atrophic rhinitis in swine, and fowl cholera (FC) in wild and domestic birds. Many strains of P. multocida express a capsule on their surfaces. The antigenic specificity of the capsule of P. multocida determines its serogroup, either A, B, D, E, or F (6, 23, 25). Interestingly, the majority of FC, HS, and atrophic rhinitis cases are caused by serogroup A strains, by serogroup B and E strains, and by serogroup D strains, respectively, suggesting that the capsule is related to the pathogenesis of the disease and to host predilection.Capsules are highly hydrated polysaccharides located external and adherent to the bacterial cell wall (28). The location of extracellular polysaccharides at the outermost surface of the cell is important because they are the first portal of entry and the last barrier to excretion of substances in and out of the cell (7). Various hypotheses have been postulated about the function of the bacterial capsule. These include protection against desiccation in the environment (19), phagocytosis (26), and the bactericidal activity of serum complement (15,32).Previous studies of the influence of the capsule on the virulence of P. multocida have used spontaneously derived acapsular variants or enzymatic removal of the capsule (1,10,11,14,17,21,27). These studies suggested that there is a correlation between the capsule and the virulence of P. multocida. However, because these strains were not genetically defined, it is not possible to ascribe definitively their phenotypes to the lack of capsule. Recently, the capsule of an HS strain of P. multocida (serotype B:2) was shown to be involved in virulence for mice by comparing an isogenic acapsular mutant to the wild type and the complemented mutant (5).We have reported previously the nucleotide sequence of the P. multoc...