Actinobacillus pleuropneumoniae, a porcine respiratory tract pathogen, has been shown to express transferrinbinding proteins and urease during infection. Both activities have been associated with virulence; however, their functional role for infection has not yet been elucidated. We used two isogenic A. pleuropneumoniae single mutants (⌬exbB and ⌬ureC) and a newly constructed A. pleuropneumoniae double (⌬ureC ⌬exbB) mutant in aerosol infection experiments. Neither the A. pleuropneumoniae ⌬exbB mutant nor the double ⌬ureC ⌬exbB mutant was able to colonize sufficiently long to initiate a detectable humoral immune response. These results imply that the ability to utilize transferrin-bound iron is required for multiplication and persistence of A. pleuropneumoniae in the porcine respiratory tract. The A. pleuropneumoniae ⌬ureC mutant and the parent strain both caused infections that were indistinguishable from one another in the acute phase of disease; however, 3 weeks postinfection the A. pleuropneumoniae ⌬ureC mutant, in contrast to the parent strain, could not be isolated from healthy lung tissue. In addition, the local immune response-as assessed by fluorescenceactivated cell sorter and enzyme-linked immunosorbent spot analyses-revealed a significantly higher number of A. pleuropneumoniae-specific B cells in the bronchoalveolar lavage fluid (BALF) of pigs infected with the A. pleuropneumoniae ⌬ureC mutant than in the BALF of those infected with the parent strain. These results imply that A. pleuropneumoniae urease activity may cause sufficient impairment of the local immune response to slightly improve the persistence of the urease-positive A. pleuropneumoniae parent strain.Actinobacillus pleuropneumoniae is the etiologic agent of porcine pleuropneumonia, a highly infectious disease of fattening pigs occurring worldwide (12). A number of putative virulence factors, such as Apx toxins, capsule, lipopolysaccharide (LPS), the ability to utilize transferrin-bound iron, and urease, have been described elsewhere (18). To date, conclusive evidence obtained by challenge experiments has been presented to confirm the role of Apx toxins and capsular material. A spontaneous Apx toxin-negative A. pleuropneumoniae strain was shown to be avirulent (14), and this result was supported later by using transposon mutagenesis (36) as well as by an isogenic A. pleuropneumoniae apxC insertion mutant (29). Also, capsule-deficient A. pleuropneumoniae strains obtained by chemical mutagenesis were shown to be attenuated (22), and this result was confirmed by reconstituting virulence properties and capsule formation upon transformation with a recombinant plasmid (39). Also, it was shown recently that the [Cu,Zn]-superoxide dismutase is not required for virulence (35). For other putative virulence factors, such as LPS (1, 3, 4), and the utilization of transferrin-bound iron (15,17,40), no conclusive challenge experiments have been performed to date. With respect to urease, data are inconclusive; ureasenegative A. pleuropneumoniae mutants hav...

No abstract

Deletions were introduced into the ureC and apxIIA genes of an Actinobacillus pleuropneumoniae serotype 2 strain by homologous recombination and counterselection. The double-mutant contains no foreign DNA, is highly attenuated, protects pigs from homologous challenge upon a single aerosol application, and facilitates the serological discrimination of immunized and infected herds.

Upon iron restriction, Actinobacillus pleuropneumoniae has been shown to express the transferrin-binding proteins TbpB and TbpA, both of which have been implied to be important virulence factors. In order to identify additional iron-regulated proteins, we cloned and analyzed the region upstream of the transferrinbinding protein genes in an A. pleuropneumoniae serotype 7 strain. We located immediately upstream of the tbpB gene two open reading frames which were 43% homologous to the neisserial ExbBD protein genes. By raising specific antibodies, we showed that ExbB is expressed under iron-limiting growth conditions only, and RT-PCR analysis revealed that the exbBD genes and the tbpB gene are transcribed on a single polycistronic mRNA. By constructing an isogenic and nonpolar exbBD mutant, we showed that the exbBD genes are required by A. pleuropneumoniae for utilization of transferrin-bound iron. Using PCR and Western blotting, we showed that the genetic organization found in A. pleuropneumoniae serotype 7 is similar in all 12 A. pleuropneumoniae serotype reference strains.

A ferrichrome receptor, FhuA, was identified in Actinobacillus pleuropneumoniae serotype 7. An isogenic mutant with a deletion in the ferrichrome uptake receptor gene (fhuA) was constructed and examined in an aerosol infection model. The disease caused by the mutant was indistinguishable from disease induced by A. pleuropneumoniae serotype 7 wild-type; an isogenic mutant lacking expression of the exbB gene that is required for the uptake of transferrin-bound iron retained the ability to utilize ferrichrome, thereby indicating that an energy-coupling mechanism involved in ferrichrome transport remains to be identified.