Progeny of horn flies feeding upon blood from animals vaccinated with BI-HS009 plus adjuvant collected on day 56 had 63% lower pupariation rate and 57% lower adult emergence than the control group (ANOVA: F (1, 6) = 8.221, P = 0.028 and F (1, 6) = 8.299, P = 0.028, respectively). The IgG titers of animals vaccinated with BI-HS009 plus adjuvant were significantly higher than those of animals vaccinated with buffer plus adjuvant only from days 42 to 112, with a peak on day 56. One of the vaccine candidate antigens, BI-HS009, was expressed in sufficient quantity for immunogenicity and efficacy trials. RT-PCR confirmed the expression of all six ORFs in adult fly RNA. Six of the ten vaccine candidate antigens were successfully expressed in P. Then, we evaluated the immunogenicity of a vaccine candidate in an immunization trial and the antigen’s effects on horn fly mortality and fecundity in an in vitro feeding assay. The expression of the 10 selected transcripts and the proteins that they encoded were investigated in adult flies by reverse transcription polymerase chain reaction (RT-PCR) and mass spectrometry, respectively. We used a reverse vaccinology approach comprising three vaccine prediction and 11 annotation tools to evaluate and rank 79,542 translated open reading frames (ORFs) from the horn fly's transcriptome, and selected 10 transcript ORFs as vaccine candidates for expression in Pichia pastoris. Horn fly control relies on insecticides however, alternative control methods such as vaccines are needed due to the fly's capacity to quickly develop resistance to insecticides, and the pressure for eco-friendly options. The horn fly, Haematobia irritans irritans, causes significant production losses to the cattle industry.
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