A Unique Capsule Locus in the Newly Designated Actinobacillus pleuropneumoniae Serovar 16 and Development of a Diagnostic PCR Assay

Abstract

Actinobacillus pleuropneumoniae causes pleuropneumonia, an economically significant lung disease of pigs. Recently, isolates of A. pleuropneumoniae that were serologically distinct from the previously characterized 15 serovars were described, and a proposal was put forward that they comprised a new serovar, serovar 16. Here we used whole-genome sequencing of the proposed serovar 16 reference strain A-85/14 to confirm the presence of a unique capsular polysaccharide biosynthetic locus. For molecular diagnostics, primers were designed from the capsule locus of strain A-85/14, and a PCR was formulated that differentiated serovar 16 isolates from all 15 known serovars and other common respiratory pathogenic/commensal bacteria of pigs. Analysis of the capsule locus of strain A-85/14 combined with the previous serological data show the existence of a sixteenth serovar-designated serovar 16-of A. pleuropneumoniae.

Keywords: Actinobacillus pleuropneumoniae; PCR; diagnostics; serovar 16.

FIG 1

Schematic representation of the A. pleuropneumoniae serovar 16 capsule locus (proposed type IV CPS), and comparison to representative loci for CPS types I to III. The CPS biosynthesis genes for serovar 16 (Sero 16) (cps16ABCDEF) are located downstream of and on the opposite strand to the capsule export genes (cpxDCBA; only cpxD is shown) as in the other loci (note that although not shown, the serovar 15 CPS biosynthetic genes are contiguous with and in the same orientation as the export genes). The relative size and location of each gene are indicated by the sizes and positions of arrows (solid gray or patterned). For type I CPS (serovars 2, 3, 6, 7, 8, 9, 11, and 13), the locus of the serovar 2 reference strain S1536 is shown. All type I CPS loci share three common genes as indicated by the arrows with hatching. For type II CPS (serovars 1, 4, 12, and 15), the locus for the serovar 1 reference strain 4074^T is shown. All type II loci share the first gene as indicated by the arrow with the dotted pattern. For type III CPS (serovars 5 and 10), the locus for the serovar 5b reference strain L20 is shown. Type III CPS loci share the three kds genes indicated by the arrows with the crosshatched pattern. The newly proposed type IV CPS locus is currently found only in serovar 16, as illustrated by the locus of the reference strain A-85/14. The locations of serovar 16-specific primers are indicated as small curved arrows above cps16C (forward primer AP16F) and cps16D (reverse primer AP16R).

FIG 2

Verification of specificity of primers for molecular identification of A. pleuropneumoniae serovar 16. An apxIV (418-bp) amplicon is detected in all 16 serovar reference strains; the serovar 16-specific amplicon (212 bp) is detected only in the serovar 16 reference strain A-85/14 and the representative clinical isolate, 151/16. Lane M contains molecular size markers (100-bp ladder). Lanes 1 to 16 contain the following strains: 1, 4074^T; 2, S1536; 3, S1421; 4, M62; 5, L20; 6, Femo; 7, WF83; 8, 405; 9, CVJ13261; 10, D13039; 11, 56153; 12, 8329; 13, N-273; 14, 3906; 15, HS143; 16, A-85/14. Lane 16a contains serovar 16 strain 151/16, as representative of all serovar 16 clinical isolates tested, and lane neg contains no DNA and is a negative control.