Temperature-sensitive mutants of Actinobacillus pleuropneumoniae induce protection in mice

Abstract

Temperature-sensitive mutants of Actinobacillus pleuropneumoniae 4074, serotype 1, were isolated after treatment with nitrosoguanidine and enrichment with penicillin and D-cycloserine. Of the four temperature-sensitive mutants evaluated in mice, one (A-1) had a tight phenotype (i.e., it ceased replication immediately after transfer to the nonpermissive temperature [37°C]) and three (1-2, 4-1, and 12-1) were coasters that continued replication for up to three generations after transfer to 37°C. The reversion frequencies ranged from 10 ؊6 to 10 ؊9 , and cutoff temperatures ranged from 33 to 35°C. No major changes were detected in the biochemical profiles; agglutination reactions; electrophoretic profiles of the lipopolysaccharides, outer membrane proteins, and hemolysin proteins; hemolytic titers; or CAMP factor reactions of the mutants and the wild-type bacteria. Groups of 3-to 5-week-old, female ICR mice were immunized intranasally with three doses of 3.5 ؋ 10 6 CFU of the mutants over 3 weeks and subsequently challenged intranasally with 5 50% lethal doses of the parental wild-type. Protection was induced by both the tight and the coaster mutants, with the 4-1 and 12-1 coasters eliciting greater protection (67 and 82%, respectively) than that induced by the A-1 tight mutant (57%). Intranasal immunization with both phenotypes induced serum antibody responses against the surface antigens and the hemolysin protein.

Construction and characterization of a live, attenuated apxIICA inactivation mutant ofActinobacillus pleuropneumoniaelacking a drug resistance marker

“…In addition to generating a potential live vaccine for A. pleuropneumoniae infections, the counter selection method developed here could also be used to generate multivalent vaccines against concomitant infections using attenuated A. pleuropneumoniae strains such as A. pleuropneumoniae HB04 À as vaccine carrier to deliver protective antigens from other organisms. 2 · 10 8 10/10 1 · 10 8 10/10 2 · 10 7 10/10 1 Mutant strain (HB04C À ) 2 · 10 9 0/10 100 1 · 10 9 0/10 2 · 10 8 0/10 Control (none) TSB broth 0/10 a Groups of 10 mice were injected intraperitoneally with 200 ll of bacterial suspension containing various quantity of A. pleuropneumoniae strains. Survival was recorded for 10 days after infection.…”

“…The construction of attenuated strains of A. pleuropneumoniae has been investigated by chemical mutagenesis, gene targeting, or transposon‐mediated approaches [6–11]. Immunization of pigs with these vaccines provided varying degrees of cross‐protection in mice or swine against mortality and lung lesions after challenge with a heterologous serotype of A. pleuropneumoniae .…”