Analyses of Dutch Haemophilus parasuis isolates by serotyping, genotyping by ERIC-PCR and Hsp60 sequences and the presence of the virulence associated trimeric autotransporters marker

Abstract

In this study, 117 isolates of Haemophilus parasuis from organs and tissues from pigs showing clinical signs, were characterised and compared with 10 H. parasuis reference strains. The isolates were subjected to the 16S rRNA gene PCR and subsequently serotyped, genotyped by 60-kDa heat shock protein (Hsp60) gene sequences, the enterobacterial repetitive intergenic consensus (ERIC) PCR and a multiplex PCR for the detection of the vtaA virulence associated trimeric autotransporter genes. Serotyping revealed the presence of 13 H. parasuis serovars. Serovars 3 and 10 were not detected, and 16 of the 117 H. parasuis isolates could not be typed by specific antisera. All isolates were positive in the 16S rRNA gene specific H. parasuis PCR. ERIC-PCR revealed a very heterogeneous pattern with 61 clusters; based on a 90% agreement. In total, 46 different Hsp60 sequence types were detected. Using 98% sequence similarity, as threshold for separation, 22 separate Hsp60 sequence clusters were distinguished. There was no correlation between H. parasuis serovars and ERIC-PCR clusters or Hsp60 sequence types, but both the ERIC-PCR and the Hsp60 sequence typing are suited as markers for H. parasuis molecular-epidemiology studies. In total, 102 H. parasuis swine isolates corresponded to the virulence associated group 1 vtaA type. The group 1 vtaA was detected in 12 different serovars. Only four of the 46 Hsp60 sequence types were not associated with the group 1 vtaA. This study shows that Dutch H. parasuis isolates from pigs with clinical signs have both a high serovar and genotypic lineage diversity. A majority of the known serovars contain the group 1 vtaA.