Brush (Badger, Franklin Park, USA) after anaesthesia with 50 O2/50 N…

Brush (Badger, Franklin Park, USA) after anaesthesia with 50 O2/50 N2O/3 halothane. In all experiments, piglets were followed clinically with special regard to signs of meningitis and arthritis. Swabs for bacteriological examination were taken daily from the oropharynx and faeces. Pigs were killed either moribund or 18 days post infection at the end of the observation period by intravenous injection of pentobarbiturate followed by exsanguination and necropsy. Tissue specimens from the central nervous system (CNS), serosae, and joints were examined bacteriologically and histologically Celecoxib [21,23].Multi Locus Sequence Typing (MLST)MethodsBacterial strains and growth conditionsBacterial isolates are described in Table 1. S. suis strains were grown on Columbia agar blood base plates (Oxoid Ltd., London, United Kingdom) containing 6 (vol/vol) horse blood. Cultures were grown in Todd-Hewitt broth (Oxoid). Escherichia coli was grown in Luria Broth (Oxoid) and plated on Luria Broth Agar (Oxoid). S. suis isolates used in this study were serotyped PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/15501003 using the slide-agglutination test [18] before they were used in the study (Table 1). Expression of three virulence markers, MRP, EF, and SLY [19,20] was confirmed for all isolates by Western blot analysis [9] using monoclonal antibodies against MRP, EF [21], or SLY [22] (Table 1).Experimental infection in pigsAll animal experiments were approved by the ethical committee of the Central Veterinary Institute of Wageningen UR in accordance with the Dutch law on animal experiments. In this study virulence of S. suis isolates was strictly defined by the outcome of experimental infections. To study virulence of S. suis serotype 1 and 9 isolates, three successive experiments were performed in pigs. Previous to infection all piglets were tested negative for S. suis carriership. In all experiments pigs were allotted to three or four groups each consisting of four or five pigs (Table 2). In the first two experiments seventeen caesarian-derived germfree piglets were housed in stainless steel incubators as described before [21]. Each piglet was infected at the age of 5 days with Bordetella bronchiseptica (3 ?107 CFU, intranasally) to predispose animals for subsequent S. suis infection. Two days later animals were infected intranasally with exponentially growing S. suis strains (1 ?106 CFU aerosol). In the third experiment, specific pathogen free (SPF) piglets with the age of 6 weeks were infected intranasally with S. suis serotype 9 isolates (1 ?109 CFU) withoutMLST was performed as described by King et al. [24]. Alternative primers for mutS were used as described previously by Rehm et al. [25]. Chromosomal DNA was isolated from stationary growing bacteria as described previously [26]. PCR reactions were performed using Taq PCR Core kit (QIAgen, Hilden, Germany) according to the manufacturer's instructions, using 5 l of diluted (1:100) chromosomal DNA as template, containing at least 350 ng of DNA. PCR products were visually inspected on 1 agarose gels containing ethidium bromide, and subsequently purified and sequenced by Macrogen (Macrogen, Seoul, Korea). Sequence data were analyzed using Lasergene software (DNAstar, Madison, USA). MLST alleles and resulting STs were assigned using the database on http://ssuis.mlst.net/. New alleles and STs were assigned by the curator of the database. Analysis of ST complexes was performed with eBURST http://www.mlst.net[27].S. suis oligoarrayA S. suis oligoarray (8 ?15 K) cont.