Henk J. Wisselink

Distribution of capsular types and production of muramidase-released protein (MRP) and extracellular factor (EF) of Streptococcus suis strains isolated from diseased pigs in seven European countries

Streptococcus suis strains (n=411), isolated from diseased pigs in seven European countries were serotyped using specific antisera against serotype 1 to 28, and were phenotyped on the basis of their muramidase-released-protein (MRP) and extracellular-factor protein (EF) production. Overall, S. suis serotype 2 appeared to be most prevalent (32%), followed by serotype 9 (20%) and serotype 1 (12%). Serotype 2 was most frequently isolated in France, Italy and Spain, whereas serotype 9 was most frequently isolated in Belgium, The Netherlands and Germany. In the United Kingdom serotypes 1 and 14 were most frequently isolated. High percentages of S. suis serotype 1, 2, 1/2 and 14 strains, isolated from tissues associated with S. suis infections such as brain, serosa, joint, heart and organs expressed the EF-protein, indicating that in these serotypes expression of EF is likely to be associated with virulence. In contrast, strains belonging to serotype 7 and 9, isolated from tissues associated with S. suis infections did not produce EF. These results strongly suggest that in the serotypes 7 and 9 EF expression is not related to virulence. More than 80% of the S. suis serotype 9 strains produced an MRP* protein, a high molecular variant of the 136kDa MRP. Expression of MRP* in serotype 9 strains is possibly associated with virulence.

Relatedness of Streptococcus suis isolates of various serotypes and clinical backgrounds as evaluated by macrorestriction analysis and expression of potential virulence traits.

ABSTRACT We evaluated the genetic diversity of Streptococcus suis isolates of different serotypes by macrorestriction analysis and elucidated possible relationships between the genetic background, expression of potential virulence traits, and source of isolation. Virulence traits included expression of serotype-specific polysaccharides, muramidase-released protein (MRP), extracellular protein factor (EF), hemolysin activity, and adherence to epithelial cells. Macrorestriction analysis of streptococcal DNA digested with restriction enzymes SmaI and ApaI allowed differentiation of single isolates that could be assigned to four major clusters, named A1, A2, B1, and B2. Comparison of the genotypic and phenotypic features of the isolates with their source of isolation showed that (i) the S. suis population examined, which originated mainly from German pigs, exhibited a genetic diversity and phenotypic patterns comparable to those found for isolates from other European countries; (ii) certain phenotypic features, such as the presence of capsular antigens of serotypes 2, 1, and 9, expression of MRP and EF, and hemolysin activity (and in particular, combinations of these features), were strongly associated with the clinical background of meningitis and septicemia; and (iii) isolates from pigs with meningitis and septicemia showed a significantly higher degree of genetic homogeneity compared to that for isolates from pigs with pneumonia and healthy pigs. Since the former isolates are considered highly virulent, this supports the theory of a clonal relationship among highly virulent strains.

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.

Detection of virulent strains of Streptococcus suis type 2 and highly virulent strains of Streptococcus suis type 1 in tonsillar specimens of pigs by PCR.

We developed a PCR assay for the rapid and sensitive detection of virulent Streptococcus suis type 2 and highly virulent S. suis type 1 in tonsillar specimens from pigs. The PCR primers were based on the sequence of the gene encoding the EF-protein of virulent S. suis type 2 strains (MRP+EF+) and highly virulent S. suis type 1 strains (MRP(s)EF+) and of the EF protein of weakly virulent S. suis type 2 strains (MRP+EF). The latter strains give rise to larger PCR products than the virulent strains of S. suis type 1 and 2. A positive control template was included in the assay to identify false negative results. The PCR was evaluated using tonsillar specimens from herds known (or suspected) to be infected and herds without an S. suis history. The results obtained with the PCR assay were compared with the results obtained with a newly developed bacteriological examination. In this bacteriological examination we were able to identify the EF-positive strains directly in the tonsillar specimens. From the 99 tonsils examined, 48 were positive in the PCR and 51 negative. All specimens from which EF-positive S. suis strains were isolated were also positive in the PCR assay. Three samples were positive in the PCR, but negative by bacteriological examination. The results demonstrated that the PCR is a highly specific and sensitive diagnostic tool for the detection of pigs carrying virulent strains of S. suis type 2 and highly virulent strains of type 1. Application of the assay may contribute to the control of S. suis infections.

Assessment of protective efficacy of live and killed vaccines based on a non-encapsulated mutant of Streptococcus suis serotype 2

The protective efficacy of a live and killed non-encapsulated isogenic mutant of Streptococcus suis serotype 2 was determined in pigs, and compared with the efficacy of the capsulated wild-type strain. SPF pigs were vaccinated twice intramuscularly at 4 and 7 weeks of age with a dose of 1 x 10(9) formalin-killed CFU of the wild-type (WT-BAC), formalin-killed non-encapsulated mutant (CM-BAC) or live non-encapsulated mutant (CM-LIVE) strain. After 2 weeks, vaccinated pigs and non-vaccinated controls were challenged intravenously with 1 x 10(7) CFU of the homologous, wild-type S. suis serotype 2 strain. Protection was evaluated by clinical, bacteriological, serological and post-mortem examinations. All pigs vaccinated with WT-BAC were completely protected against challenge with the homologous serotype. Pigs vaccinated with CM-BAC were partially protected. Although all pigs vaccinated with CM-BAC survived the challenge, four out of five pigs developed clinical signs of disease for several days. Compared to the WT-BAC and CM-BAC, the CM-LIVE vaccine was less protective. Two out of five pigs vaccinated with CM-LIVE died in the course of the experiment and all of them developed specific clinical signs of disease for several days. The protective efficacy of the vaccines could be associated with serum antibody titers. Antibody titers against cells of wild-type and non-encapsulated mutant strains as well as against muramidase-released proteins (MRP) were high in pigs vaccinated with WT-BAC and CM-BAC. Pigs vaccinated with CM-LIVE showed lower antibody titers. Antibody titers against purified capsular polysaccharides (CPS) of S. suis serotype 2 were only found in pigs vaccinated with WT-BAC. These findings indicate that CPS and other bacterial components of WT-BAC are probably essential for full protection against homologous challenge.

Genetic diversity of Streptococcus suis isolates as determined by comparative genome hybridization

BackgroundStreptococcus suis is a zoonotic pathogen that causes infections in young piglets. S. suis is a heterogeneous species. Thirty-three different capsular serotypes have been described, that differ in virulence between as well as within serotypes.ResultsIn this study, the correlation between gene content, serotype, phenotype and virulence among 55 S. suis strains was studied using Comparative Genome Hybridization (CGH). Clustering of CGH data divided S. suis isolates into two clusters, A and B. Cluster A isolates could be discriminated from cluster B isolates based on the protein expression of extracellular factor (EF). Cluster A contained serotype 1 and 2 isolates that were correlated with virulence. Cluster B mainly contained serotype 7 and 9 isolates. Genetic similarity was observed between serotype 7 and serotype 2 isolates that do not express muramidase released protein (MRP) and EF (MRP-EF-), suggesting these isolates originated from a common founder. Profiles of 25 putative virulence-associated genes of S. suis were determined among the 55 isolates. Presence of all 25 genes was shown for cluster A isolates, whereas cluster B isolates lacked one or more putative virulence genes. Divergence of S. suis isolates was further studied based on the presence of 39 regions of difference. Conservation of genes was evaluated by the definition of a core genome that contained 78% of all ORFs in P1/7.ConclusionsIn conclusion, we show that CGH is a valuable method to study distribution of genes or gene clusters among isolates in detail, yielding information on genetic similarity, and virulence traits of S. suis isolates.

Environmentally regulated genes of Streptococcus suis: identification by the use of iron-restricted conditions in vitro and by experimental infection of piglets.

The identification of environmentally regulated genes of Streptococcus suis by the use of iron-restricted conditions in vitro and by experimental infection of piglets is described. Eighteen unique iron-restriction-induced (iri) genes and 22 unique in-vivo-selected (ivs) genes of Strep. suis were found. None of the ivs genes was exclusively expressed in vivo. Four iri genes were identical to four clones selected in piglets. Two ivs genes were similar to genes for putative virulence factors. One of these ivs genes was identical to the epf gene of virulent Strep. suis serotype 2 strains and the other showed homology to a gene encoding a fibronectin-binding protein of Streptococcus gordonii. Two additional ivs genes showed homology to environmentally regulated genes previously identified by using an in vivo expression technology (IVET) selection system in other bacterial species. One of these showed similarity to the agrA gene of Staphylococcus aureus, a key locus involved in the regulation of numerous virulence proteins. The promoter selection system described in this paper has been successfully used for the identification of many environmentally regulated genes potentially involved in the pathogenesis of Strep. suis infections in piglets.

Virulence Markers of Streptococcus suis Type 1 and 2

Streptococcus suis infections are a common cause of meningitis, septicemia, arthritis, sepsis and sudden death in young pigs and meningitis in humans. During the last few years, S. suis infections have become a major problem in almost all countries with an intensive pig industry. Attempts to control the disease are hampered by the lack of effective vaccines and suitable diagnostics. Until now 32 different serotypes of S. suis have been described, but the serotypes 1 and 2 are isolated most frequently. Within serotypes strains can differ in virulence.

Characterization of virulence of the Streptococcus suis serotype 2 reference strain Henrichsen S 735 in newborn gnotobiotic pigs

Strain Henrichsen S 735 (NCTC 10234) of Streptococcus suis serotype 2 reference and three other such strains (strains S 4005, S 3921 and T 141) were tested for virulence by inoculating pigs intranasally and intravenously. The taxonomical properties of each strain were determined. Phenotypes were determined by Western blotting based on MRP and EF protein expression and genotypes were determined by Southern hybridization analysis of the mrp and epf genes. Reference strain S 735 and strain S 3921 produced the 136 kDa MRPh and a 180 kDa form of EF, and hence these strains belong to the MRP + EF phenotype. In accordance with previous experiments with this phenotype, strains S 735 and S 3921 appeared to be only weakly virulent for newborn gnotobiotic pigs. Strain S 4005 produced the 136 kDa MRP and the 110 kDa form of EF, hence it belongs to the MRP + EF + phenotype. This strain was highly virulent for pigs. Strain T 141 did not produce MRP or EF, and hence belongs to the MRP-EF- phenotype. It was nonvirulent for pigs. The route of inoculation did not influence the frequency or severity of clinical signs of disease or lesions, which demonstrated that the 110 kDa EF is not essential during invasion. Southern blot analysis showed that all four S. suis type 2 strains contain sequences that are homologous to the epf and mrp genes. For studies on pathogenesis of S. suis type 2 infections in pigs, we recommend the use of strains that have been tested in a standardized pig model and that belong to the MRP + EF + phenotype, such as strain S 4005.

Virulence of streptococcus suis type 2 for mice and pigs appeared host-specific

A murine model for Streptococcus suis infection in pigs was validated by inoculating groups of 5 BALB/c and 5 CF1 mice with 10(7) CFU/ml of 13 different S. suis serotype 2 strains. The pathogenicity of these strains had been established in a standardized pig model of S. suis infection using one-week-old gnotobiotic pigs. We inoculated groups of mice intraperitoneally with 4 strains that were highly virulent for pigs and belonged to the phenotype MRP+EF+, with 4 strains, that were weakly virulent for pigs and belonged to the phenotype MRP+EF+, and with 5 strains that were non-virulent for pigs and belonged to phenotype MRP-EF-. The S. suis strains that were highly virulent for pigs caused high morbidity and an intermediate mortality in mice, the S. suis strains that were weakly virulent for pigs caused high morbidity but low mortality, and the strains that were non-virulent for pigs, induced highest morbidity and mortality. These results were comparable in both breeds of mice. In contrast to the pathology of S. suis infection in pigs with specific lesions, lesions in mice were histologically often characterized as non-specific, i.e., necrotizing encephalitis and focal or diffuse hepatitis sometimes with abscesses. Irrespective of breed (BALB/c vs. CF1), the murine model used for S. suis infection was incompatible with the pig model. This indicates that virulence of S. suis type 2 for mice and pigs is host-specific. Therefore, we regard the presently available murine models unsuitable for studying S. suis infections in pigs.