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Keywords:

  • dihydropteroate synthase;
  • Haemophilus parasuis;
  • representational difference analysis;
  • respiratory pathogen;
  • sulfonamide resistance

Abstract

Aims:  Identification of genes differentially present in Haemophilus parasuis serovar 2 by representational difference analysis (RDA).

Methods and Results:  Bacterial genomic DNA was extracted, cleaved with Sau3AI and ligated to oligonucleotide adapter pair. The optimal tester (H. parasuis serovar 2)/driver ratio (H. parasuis serovars 1, 3 and 5) for the hybridization was established and the mixture was hybridized, and amplified by PCR. The products were cloned and transformed into Escherichia coli TOP10 cells and checked for specificity by Southern blotting analysis. The RDA subtractive technique yielded six bands ranging from 1500 to 200 bp, which were cloned into pCR II-TOPO vector and 40 clones were analysed. A fragment of 369 bp was specific for H. parasuis serovar 2, and showed 99% homology to sulI gene encoding for dihydropteroate synthase (dhps). The dhps gene conferring sulfonamide resistance was detected in H. parasuis serovar 2 but was absent in serovars 1, 3, 5 and in most of the Actinobacillus pleuropneumoniae serotypes (except serotype 7).

Conclusion: sulI allele of dihydropteroate synthase has been identified in H. parasuis serovar 2 by RDA technique.

Significance and Impact of the Study:  The RDA technique seems to be an useful method for the identification of genes that are differentially present in H. parasuis, a respiratory pathogen of veterinary interest.