Oral immunization of mice with attenuated Salmonella typhimurium vaccine expressing transferrin-binding protein A (TbpA) of Haemophilus parasuis

ABSTRACT

Background: Haemophilus parasuis is the etiological agent responsible for causing Glässers disease in pigs, which aremajor 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 arecapable of binding to transferrin, and an impairment of iron uptake mechanisms is likely to induce virulence. For thisreason, these proteins can be useful as a candidate target for H. parasuis vaccination. Also, the live attenuated SalmonellaTyphimurium 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 neurtophilpassage method. By the passage, the ability of the neutrophil-adapted isolate to utilize d-xylose was lost, while the abilityof 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 PCRusing primers specific to the endpoints of the targeted region. TbpA fragment was amplified by PCR from genomic DNAof H. parasuis serotype 5. To construct TbpA expression plasmids, tbpA was subcloned downstream from the β-lactamasesignal sequence in the multicopy asd+ pYA3493 vector. This plasmid was subsequently electrotransformed into attenuatedS. Typhimurium. The 636bp fragment of the tbpA gene of H. parasuis in attenuated S. Typhimurium was amplified by PCRand the in-frame fusion of the tbpA was confirmed by nucleotide sequencing. The used this strain with Asd+ balanced-lethalplasmid 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...(AU)