The cps genes of Streptococcus suis serotypes 1, 2, and 9: development of rapid serotype-specific PCR assays

Abstract

We developed three type-specific PCR assays for the rapid and sensitive detection of Streptococcus suis serotype 1 (plus 14), serotype 2 (plus 1/2), and serotype 9 strains in tonsillar specimens from pigs. The PCR primers were based on the sequences of type-specific capsular genes of S. suis serotype 1, 2, and 9 strains. We recently characterized a major part of the capsular biosynthesis (cps) locus of S. suis serotype 2. Here we extended these studies and characterized major parts of the cps loci of S. suis serotypes 1 and 9. Type-specific genes were identified by cross-hybridization experiments between the individual cps genes and chromosomal DNAs from the 35 different serotypes. Four genes of S. suis serotype 1 specifically hybridized with serotype 1 and 14 strains only. Five genes of S. suis serotype 2 specifically hybridized with serotype 2 and 1/2 strains only, and two genes of S. suis serotype 9 specifically hybridized with serotype 9 strains. Until now rapid and sensitive diagnostic tests were available only for pathogenic strains of serotype 2 and highly pathogenic strains of serotype 1. The serotype-specific PCR assays can therefore be useful tools for the identification of serotype 1, 14, 2, 1/2, and 9 strains both for diagnostic purposes and in epidemiological and transmission studies. Therefore, these tests may facilitate control and eradication programs.

FIG. 1

The cps2, cps1, and cps9 gene clusters of S. suis serotypes 2, 1, and 9. (A) Genetic organization of the cps2 gene cluster. The large arrows represent potential ORFs. Gene designations are indicated below the ORFs. Identically filled arrows represent ORFs which showed homology. The small closed arrows indicate the position of the potential promoter sequences. , position of the potential transcription regulator sequence. (B) Physical map and genetic organization of the cps1 gene cluster. Restriction sites are as follows: E, EcoRI; EV, EcoRV; H, HindIII. The DNA fragments cloned in the various plasmids are indicated. The open arrows represent potential ORFs. As a result of the construction of the plasmids, cps1E is incomplete at its 5′ end and cps1K is incomplete at its 3′ end. (C) Physical map and genetic organization of the cps9 gene cluster. Restriction sites are as follows: B, BamHI; H, HindIII; X, XbaI. The DNA fragments cloned in the various plasmids are indicated. The open arrows represent potential ORFs. As a result of the construction of the plasmids, cps9D is incomplete at its 5′ end and cps9H is incomplete at its 3′ end.

FIG. 2

(A) Ethidium bromide-stained agarose gel showing PCR products obtained with chromosomal DNA of S. suis strains belonging to serotypes 1, 2, 1/2, 9, and 14 and cps2J, cps1I, and cps2H primer sets. (I) cps1I primers; (II) cps2J primers; (III) cps9H primers. Lanes 1 to 3, serotype 1 strains; lanes 4 to 6, serotype 2 strains; lanes 7 to 9, serotype 1/2 strains; lanes 10 to 12, serotype 9 strains; lanes 13 to 15, serotype 14 strains; lane 16, negative control, no DNA, cps2J primers; lanes M, molecular size marker (bacteriophage lambda digested with EcoRI and HindIII). (B) Ethidium bromide-stained agarose gel showing PCR products obtained with tonsillar specimens collected from pigs carrying S. suis serotype 2, serotype 1, or serotype 9 strains and cps2J, cps1I, and cpsH primer sets. Bacterial DNA suitable for PCR was prepared by using the multiscreen methods described previously (21). (I) cps1I primers; (II) cps2J primers; (III) cps9H primers. Lanes 1 to 3, PCR products obtained with tonsillar specimens collected from pigs carrying S. suis serotype 1 strains (as detected by regular bacteriological examination); lanes 4 to 6, PCR products obtained with tonsillar specimens collected from pigs carrying S. suis serotype 2 strains; lanes 7 to 9, PCR products obtained with tonsillar specimens collected from pigs carrying S. suis serotype 9 strains; lanes 10 to 12, PCR products obtained with chromosomal DNA from serotype 9, 2, and 1 strains, respectively; lane 13, negative control, no DNA, cps2J primers; lanes M, molecular size marker (bacteriophage lambda digested with EcoRI and HindIII).