Multiplex PCR assays for simultaneous detection of six major serotypes and two virulence-associated phenotypes of Streptococcus suis in tonsillar specimens from pigs

Abstract

Multiplex PCR assays for the detection and identification of various Streptococcus suis strains in tonsillar specimens from pigs were developed and evaluated. In two separate reactions, five distinct DNA targets were amplified. Three targets, based on the S. suis capsular polysaccharide (cps) genes specific for serotypes 1 (and 14), 7, and 9, were amplified in multiplex PCR I. Two other targets, based on the serotype 2- (and 1/2-) specific cps gene and the epf gene, encoding the EF proteins of virulent serotype 2 and highly virulent serotype 1 strains, were amplified in multiplex PCR II. To identify false-negative results, firefly luciferase (luc) DNA and primers based on the luc gene were included in the assay. The multiplex PCR assays were evaluated with tonsillar specimens from pigs infected with S. suis strains. The results obtained with the PCR assays were compared with the results obtained with a bacteriological examination. Most (94%) of the results obtained with multiplex PCR assays were confirmed by the bacteriological examination. The PCR method seems to be more sensitive compared to the bacteriological method, since the remaining 6% of the samples were positive by PCR and negative by bacteriological examination. These results indicate that the PCR method is highly specific for the detection of S. suis strains most frequently involved in clinical disease in infected pig herds. The serotypes found by PCR in tonsillar specimens from diseased pigs were compared with the serotypes of the strains isolated from the affected tissues of the same pigs. The results showed that there is significant association between carriership and clinical illness for S. suis serotype 9 and EF-positive serotype 2 strains and not for serotype 7 and EF-negative serotype 2 (or 1/2) strains.

FIG. 1.

PCR products obtained in multiplex PCR I (A) or multiplex PCR II (B) with purified chromosomal DNA (1 ng) of eight S. suis strains spiked with 10 fg of pGL2-Basic vector. Samples were separated on a 2% agarose gel stained with ethidium bromide. Lane designations are indicated. S. suis serotype 1/2, strain 5209 (lane 1); S. suis serotype 1, strain 5210 (lane 2); S. suis serotype 2 (EF*-positive), strain 5211 (lane 3); S. suis serotype 7, strain 5216 (lane 4); S. suis serotype 9, strain 5218 (lane 5); S. suis serotype 14, strain 5223 (EF*-positive) (lane 6); S. suis type 1 (EF-positive), strain 6388 (lane 7); S. suis type 2 (EF-positive), strain 4005 (lane 8); negative control (without S. suis DNA) (lane 9); mixture of chromosomal DNA (1 ng) of strains in lanes 1 to 8 (lane 10). Lane MW contains DNA molecular size markers (0.019 to 1.11 kbp; Roche); the sizes (in base pairs) of the PCR products are indicated on the left.

FIG. 2.

Sensitivity of multiplex PCR I (A) and II (B) on a mixture of chromosomal DNAs of S. suis serotype 1, 7, and 9 (A) or on an EF-positive S. suis serotype 2 strain (B). Samples were separated on a 2% agarose gel stained with ethidium bromide. The amount of target DNA tested in the PCR assays is indicated above the lanes. All samples contained 10 fg of positive control DNA. Lane MW contains DNA molecular size markers; the sizes (in base pairs) of the PCR products are indicated on the left.

FIG. 3.

PCR products in multiplex PCR I (A) and multiplex PCR II (B) obtained directly on tonsillar specimens collected from diseased pigs carrying serotype 1 (and 14), 2 (and 1/2), 7, and 9 and EF-positive S. suis strains as detected by bacteriological examination. PCR products were obtained from tonsillar specimens collected from pigs carrying S. suis serotype 1/2 (lane 1); S. suis serotype 7 (lane 2); S. suis serotype 9 (lane 3); S. suis serotype 7 and 9 strains (lane 4); S. suis serotype 7 and 14 strains (lane 5); S. suis serotype 1/2 and 9 strains (lane 6); S. suis serotype 2 (EF-positive phenotype) and 7 strains (lane 7); S. suis serotype 2 (EF-positive phenotype) and 9 strains (lane 8); and S. suis serotype 1/2, 7, and 9 strains (lane 9). All samples contained 10 fg of positive control DNA. Lane MW contains DNA molecular size markers; the sizes (in base pairs) of the PCR products are indicated on the left.