Bacteriology division, Animal and Plant Quarantine Agency

ABSTRACT Background: Haemophilus parasuis is the etiological agent responsible for causing Glässer's disease in pigs, which are major respiratory pathogens that cause large economic losses in the pig industry worldwide. H. parasuis obtains transferrinbound iron by expressing two surface receptors, transferrin-binding protein A and B (TbpA and B). The TbpA and B are capable of binding to transferrin, and an impairment of iron uptake mechanisms is likely to induce virulence. For this reason, these proteins can be useful as a candidate target for H. parasuis vaccination. Also, the live attenuated Salmonella Typhimurium expressing recombinant antigens from other pathogens are attractive vaccine vectors. Materials, Methods & Results: In this study, we constructed attenuated S. Typhimurium vaccine strain by porcine neurtophil passage method. By the passage, the ability of the neutrophil-adapted isolate to utilize d-xylose was lost, while the ability of the strain to ferment trehalose was delayed after 2 or more days of the culture. The aspartate β-semialdehyde dehydrogenase (asd) gene was eliminated from S. Typhimurium by one-step PCR. Deletion of asd region was confirmed by PCR using primers specific to the endpoints of the targeted region. TbpA fragment was amplified by PCR from genomic DNA of H. parasuis serotype 5. To construct TbpA expression plasmids, tbpA was subcloned downstream from the β-lactamase signal sequence in the multicopy asd + pYA3493 vector. This plasmid was subsequently electrotransformed into attenuated S. Typhimurium. The 636bp fragment of the tbpA gene of H. parasuis in attenuated S. Typhimurium was amplified by PCR and the in-frame fusion of the tbpA was confirmed by nucleotide sequencing. The used this strain with Asd + balanced-lethal plasmid pYA3493 vector to specify recombinant TbpA antigen, conserved immunogenic region of H. parasuis. Expression of the TbpA protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The size of TbpA protein was estimated at about 30kDa. Mice were administered orally in order to evaluate protective efficacy of this vaccine strain against H. parasuis serotype 5. For efficacy test, female ICR mice (5 weeks old) were orally injected, intraperitoneally challenged with a lethal dose (4X10 5 CFU/mouse) of H. parasuis serotype 5, and examined the survival rates compared with vaccination and non-vaccination group. The experiment was terminated two weeks post-challenge. The live attenuated S. Typhimurium conserved pYA3493-TbpA antigen vaccine protected 40% of immunized mice against challenge of H. parasuis serotype 5. This result suggested that the live attenuated Salmonella Typhimurium vaccine expressing TbpA might be protection for Glässer's disease outbreaks caused by H. parasuis. Discussion: This paper has shown protected mice that attenuated S. Typhimurium strain using pYA3493 expresses TbpA antigen against H. parasuis. Vaccination using bacterins is an efficient way to control outbreaks of Glässers disease, but significant variability has been reported. Therefore, the development of a new vaccine system to prevent Glässers disease using pYA3493-TbpA will avoid the disadvantages of the currently used vaccines. We need further works to enhance protection rate and to determine the potential of the vaccine in pigs