Association of Actinobacillus pleuropneumoniae capsular polysaccharide with virulence in pigs

Abstract

The capsular polysaccharide (CP) of Actinobacillus pleuropneumoniae is required for virulence of the bacteria in swine. However, a molecular investigation of whether the type or quantity of CP affects A. pleuropneumoniae virulence has not been reported. To initiate this investigation, a DNA region downstream of conserved genes required for CP export in A. pleuropneumoniae serotype 1 was cloned and sequenced. Three open reading frames, designated cps1A, cps1B, and cps1C, were identified that had amino acid homology to bacterial carbohydrate biosynthesis genes. A kanamycin resistance cassette (Kan(r)) was inserted into a 750-bp deletion spanning cps1AB or into a 512-bp deletion in cps1B only, and the constructs were cloned in a suicide vector. The Kan(r) gene was then transferred into the chromosome of strain 4074 by homologous recombination to produce strain 4074Deltacps1N and strain 4074Deltacps1B, respectively. Strain 4074Deltacps1N produced no detectable CP, but strain 4074Deltacps1B made 15% of the serotype 1 CP made by the parent strain, 4074, as determined by enzyme-linked immunosorbent assay and precipitation of free CP. The cps1ABC genes of strain 4074 and the cps5ABC and cps5ABCDE genes of serotype 5a strain J45 were cloned into the shuttle vector pLS88 and electroporated into 4074Deltacps1N to produce 4074Deltacps1N(pABcps101), 4074Deltacps1N(pJMLcps53), and 4074Deltacps1N(pABcps55), respectively. Strain 4074Deltacps1N(pABcps101) produced about 33% of the serotype 1 CP produced by strain 4074. Strains 4074Deltacps1N(pJMLcps53) and 4074Deltacps1N(pABcps55) produced serotype 5a CP in similar quantity or in fourfold excess, respectively, to that produced by strain 4074. With intratracheal challenge in pigs at similar dosages, the order of virulence of strains producing serotype 1 CP (assessed by mortality, lung consolidation, hemorrhage, and fibrinous pleuritis) was the following: strain 4074 > strain 4074Deltacps1N(pABcps101) > or = strain 4074Deltacps1N > strain 4074Deltacps1B. Strain 4074Deltacps1N(pJMLcps53) was less virulent than strain 4074Deltacps1N(pABcps55). However, both strains produced serotype 5a CP in similar or greater quantities than was observed for production of serotype 1 CP by the parent strain, 4074, but were less virulent than the parent strain. Therefore, the amount of serotype 1 or 5a CP produced by isogenic strains of A. pleuropneumoniae correlated with the virulence of the bacteria in pigs. However, virulence was also influenced by the type of CP produced or by its mechanism of expression.

FIG. 1.

Physical map of pGSAp11 insert from A. pleuropneumoniae strain 4074. Solid-fill arrows indicate the location and direction of transcription of the two complete open reading frames (cps1AB), identified by sequencing. Solid-unfilled arrows indicate the location and direction of transcription of the incomplete cpsC and cpxD genes located on this DNA fragment. Shaded rectangles indicate the regions deleted by mutagenesis. Line arrows indicate locations of the primers used in PCR.

FIG. 2.

Deletion of cps1AB in strain 4074Δcps1N. (A) The primers CPS1U2 and CPS1L3 were used to amplify the region deleted by mutagenesis of cps1AB. (B) The primers CPS1U3 and CPS1L4 were used to amplify the region not deleted by mutagenesis. The strains used were the serotype 5a knockout mutant strain J45-100 as a negative control (lanes 2), the serotype 1 parent strain 4074 (lanes 3), and the serotype 1 knockout mutant strain 4074Δcps1N (lanes 4).

FIG. 3.

Qualitative determination of CP production. Latex beads conjugated to IgG specific for serotype 1 CP was used. The parent strain, 4074, producing serotype 1 CP, agglutinated with the beads, while the nonencapsulated mutant 4074Δcps1N did not.

FIG. 4.

Bactericidal activity of precolostral calf serum for strains 4074, 4074Δcps1N, and J45-100. The percent viability of each strain was evaluated 0 and 60 min after incubation at 37°C with 25% precolostral calf serum. Each data point represents the mean for three separate experiments performed in duplicate.

FIG. 5.

Qualitative determination of CP production by latex beads conjugated to IgG specific for serotype 5 CP. Strains 4074 and 4074Δcps1N did not produce serotype 5 CP and did not agglutinate the beads, while the serotype 5 CP-producing chimeric strains 4074Δcps1N(pABcps55) and 4074Δcps1N(pJMLcps53) did agglutinate.