Detection and identification of Actinobacillus pleuropneumoniae serotype 5 by multiplex PCR

Abstract

Serotyping of Actinobacillus pleuropneumoniae is based on detection of the serotype-specific capsular antigen. However, not all isolates can be serotyped, and some may cross-react with multiple serotyping reagents. To improve sensitivity and specificity of serotyping and for early detection, a multiplex PCR assay was developed for detection of A. pleuropneumoniae and identification of serotype 5 isolates. DNA sequences specific to the conserved export and serotype-specific biosynthesis regions of the capsular polysaccharide of A. pleuropneumoniae serotype 5 were used as primers to amplify 0.7- and 1.1-kb DNA fragments, respectively. The 0.7-kb fragment was amplified from all strains of A. pleuropneumoniae tested with the exception of serotype 4. The 0.7-kb fragment was not amplified from any heterologous species that are also common pathogens or commensals of swine. In contrast, the 1.1-kb fragment was amplified from all serotype 5 strains only. The assay was capable of amplifying DNA from less than 10(2) CFU. The A. pleuropneumoniae serotype 5 capsular DNA products were readily amplified from lung tissues obtained from infected swine, although the 1.1-kb product was not amplified from some tissues stored frozen for 6 years. The multiplex PCR assay enabled us to detect A. pleuropneumoniae rapidly and to distinguish serotype 5 strains from other serotypes. The use of primers specific to the biosynthesis regions of other A. pleuropneumoniae serotypes would expand the diagnostic and epidemiologic capabilities of this assay.

FIG. 1

Agarose gel electrophoresis of PCR products from A. pleuropneumoniae serotype 5 genomic DNA. Lanes: 1, 1-kb DNA ladder; 2, cps primers A and B; 3, cpx primers C and D; 4, primers A, B, C, and D.

FIG. 2

Agarose gel electrophoresis of PCR products amplified from whole cells of serotypes 1 through 6 at a MgCl₂ concentration of 5 mM. Lanes: 1, 1-kb ladder; 2 through 7, PCR products from serotypes 1 through 6, respectively, amplified with primers A, B, C, and D.

FIG. 3

Agarose gel electrophoresis of PCR products amplified from whole cells of encapsulated and nonencapsulated serotype 5 strains. Lanes: 1, 1-kb ladder; 2, encapsulated strain K17; 3, chemically induced nonencapsulated mutant K17-C; 4, nonencapsulated recombinant mutant J45-100 containing a large deletion in cps; 5, chemically induced nonencapsulated mutant J45-C; 6, encapsulated strain J45.

FIG. 4

Agarose gel electrophoresis of bacterial samples of serotypes 1 through 12 at a MgCl₂ concentration of 2 mM. Lanes: 1, 1-kb DNA ladder; 2 through 7, PCR products from serotypes 1 through 6, respectively; 8 through 14, PCR products from serotypes 5 and 7 through 12, respectively; 15, 1-kb DNA ladder. All products were amplified with primers A, B, C, and D.

FIG. 5

Agarose gel electrophoresis of PCR products of bacterial samples of respiratory swine pathogens amplified with primers A, B, C, and D. Lanes: 1, A. pleuropneumoniae serotype 5; 2, A. suis; 3, B. bronchiseptica; 4, H. parasuis; 5, P. multocida; 6, S. suis; 7, 1-kb DNA ladder.

FIG. 6

(a) Agarose gel electrophoresis of PCR products from bacterial samples used for Southern hybridizations. Lanes: 1, 1-kb ladder; 2, serotype 2; 3, serotype 4; 4, serotype 5; 5, S. suis; 6, P. multocida; 7, H. parasuis. (b) Southern blot of PCR products from bacterial samples hybridized with a cpx probe. Lanes: 1, serotype 2; 2, serotype 4; 3, serotype 5; 4, S. suis; 5, P. multocida; 6, H. parasuis.

FIG. 7

Agarose gel electrophoresis of PCR products from lung tissue samples taken from swine that had been infected with serotype 5. Lanes and strain numbers: 1, 204; 2, 205; 3, 207; 4, 209; 5, 211; 6, 215; 7, 216; 8, 220; 9, 221; 10, 223; 11, 228. Lane 12, 1-kb DNA ladder.