PhD Thesis defense by Verena Blume Serrano at the Faculty of Veterinary Medicine of the Complutense University of Madrid

June 16^th, 2010

Streptococcus suis (S. suis) is an important swine pathogen, being widespread in the pig population worldwide and endemic in most pig-rearing countries of the world. Besides of it sanitary and economic impact, S. suis is also a zoonotic agent responsible for severe clinical disease in human. These facts have determinate that nowadays this pathogen is considered one of the emerging diseases both in Veterinary and Human Medicine.

There is a lack of knowledge about S. suis population structure and also about the virulence potential of determinate clones to cause disease in swine. In order to improve the comprehension of these aspects, in the present study we carried out a phenotypic typing (biochemical profile, serotyping and study of virulence-associated proteins MRP, EF and SLY) and also molecular typing (pulse field gel electrophoresis [PFGE] and multilocus sequence type [MLST]) of Streptococcus suis serotypes 2 and 9 isolates recovered in Spain from diseased and clinically healthy pigs. Results of the phenotypic typing allowed us to study S. suis biochemical diversity, to compare expression of the virulence-associated proteins between serotypes 2 and 9, and also between clinical isolates and tonsilar isolates within the same serotype. Moreover, we studied virulence-associated genes diversity. Molecular typing let us study S. suis genetic diversity and genetic similarity, and also determining the existence of a prevalent or a few prevalent clones responsible for a large proportion of clinical cases. In addition, MLST analysis data were used to compare our S. suis isolates and S. suis circulating in Europe.

This typing study was focused on S. suis type 2 and type 9 because they are the most commonly associated with swine disease in most countries worldwide. All the isolates (n=244) examined in the present study were properly identified by conventional test and by PCR, and a relatively high biochemical diversity was observed between S. suis isolates. Serotyping confirm the predominance of S. suis serotype 2 from diseased pigs in Spain as well as recently reported increase in the prevalence of serotype 9 from diseased pigs. All the isolates were also tested for the production of the virulence-associated proteins MRP, EF and SLY, and serotypes 2 and 9 expressed different virulence phenotypes, being the phenotypes MRP+EF+SLY+ and MRP+EF+SLY- the most frequently detected in serotype 2 strains, while the phenotypes MRP-EF-SLY+ and MRP-EF-SLY- were the predominant phenotypes among the serotype 9 strains. Within each serotype, no differences in the expression of virulence associated phenotypes were found between the strains isolated from tonsils and those isolated from clinical samples. The PCR assays allowed the amplification of the virulence-associated genes in that strains of both serotypes where the respective proteins were detected by Western blotting or microtitulation. Nevertheless, the respective gene was also detected in most of the SLY and MRP negative strains, but not in the EF negative strains. These genes were sequenced and they showed between 90% and 100% homology with GenBank sequences. We determinated an mrp variant with a 90bp deletion.

The PFGE typing of S. suis 244 isolates indicated a substantial genetic diversity among the S. suis isolates analyzed. Strains of serotype 2 and serotype 9 exhibited similar genetic diversity. S. suis isolates grouped in two major PFGE clusters at 65% genetic similarity. S. suis isolates of serotype 9 exhibited a lower genetic relatedness than isolates of serotype 2. Despite the genetic diversity observed, some of the PFGE profiles were more frequently isolated than others from clinical cases and they exhibited a wider distribution over herds. Clinical and tonsilar isolates were distributed through out the dendogram and they were genetically related. Several isolates of these prevalent PFGE profiles were subjected to MLST analysis. Nearly all serotype 2 isolates were assigned to ST1 complex. Only one isolate was assigned to a new allelic profile ST124. Serotype 9 isolates were assigned to two new allelic profiles, ST123 and S125. ST123 resulted in a new gki allele description, while ST125 resulted in a new allelic combination. Based on the eBURST results serotypes 2 and 9 isolates were assigned to the ST1 and ST61 complexes, respectively. The ST1 complex represents a highly successful clone that has spread worldwide and typically express the phenotype MRP+EF+SLY+ which was also the most frequent phenotype expressed by serotype 2 isolates in the present study. Considering that Spain imports pigs from countries of Central Europe, it was expected that isolates of serotype 9 would belong to the prevalent ST87 complex circulating in Central Europe. However, analysis of the allelic profiles of the ST123 and ST125 genotypes indicated that they come from two Spanish serotype 9 isolates of the ST61 complex detected in Spain before 2002. Therefore, genotypes ST123 and ST125 are distinct from the widespread European ST87 complex. This result suggest that serotype 9 isolates in Spain are related to the emergence and dissemination of the ST61 clone among the pig population. These genotypes differ also from isolates of the ST87 clone with regards the expression of virulence related proteins. Most serotype 9 isolates examined in the present study did not express MRP*, while the majority of European isolates express this protein.