Progress 06/01/04 to 05/31/05
Outputs
Objectives were to (1) express and purify a recombinant form of a newly discovered horn fly salivary protein (Hematollogen - HEM) in sufficient quantities for immunization and testing of cattle and (2) immunize cattle with the new recombinant protein and determine effects of immunization on the ability of the female fly to ingest blood and develop eggs. Quantities of Hematollogen sufficient for vaccination were produced in transformed E. coli and the fusion protein isolated only from the bacterial inclusion bodies. About 0.5-1.0 mg of fusion proteins (>95% pure) was obtained from one gram of fresh bacterial cells that were harvested from 300-400 ml bacterial culture. Immunization of Holstein calves (n = 16) was initiated, with 4 animals randomly allocated to each of the following treatment groups: 1. OV-A (ovalbumin control for protein); 2. TS9 (evaluation of a new thrombostasin isoform); 3. HEM (the new salivary protein); 4. TB8 (a thrombostasin isoform used
successfully from trials in 2003). There was variation among calves in the total serum IgG antibody titers relative to their specific vaccine antigen, perhaps accounting for a lack of statistically significant difference associated with treatment (ANOVA, n=15, p=0.469), in spite of a trend of HEM > TB8 > TS9 > OVA. Following exposure to horn fly saliva - during experimental testing followed by 3 weeks of field exposure - calves immunized with OVA and HEM showed low antibody response to TB8 protein, whereas calves immunized with TS9 protein recognized TB8 antigen equally as well as TS9 antigen Volumes of blood consumed in a 20 min feeding time were significantly less for flies feeding on calves immunized with all three salivary antigens when compared to flies feeding on OVA-immunized control calves (ANOVA, n=855; Trt, p= 0.001). There were also significant effects on blood volume consumed associated by group (p=0.002) and with calf (p=0.023). There were no significant differences in
blood volumes associated with sex of the fly (p=0.204) or feeding time (p=0.680). The percent of live flies following 24, 48 or 72 hrs of continuous exposure to immunized calves appeared to be lower when exposed to calves immunized with salivary proteins compared to OVA, but was greatest, and statistically significant, for feeding on HEM- immunized calves (ANOVA, n=16, p=0.034). Patents: (1) Antithrombin Nucleotides and Protein from Horn Fly. Chinese Patent No. ZL99811525.8. Issued September 22, 2004; (2) Antithrombin Nucleotides and Proteins from Horn Fly. Mexican Patent Application No. PA/a/2001/001849. Filed November 17, 2004; (3) Protein From Horn Fly Saliva That Disrupts Hemostasis. Filed November 23, 2004 - Patent Pending.
Impacts
This newly-described salivary factor could prove important in the development of a polyvalent anti-feeding vaccine directed against the horn fly. It differs in structure from thrombostasin, another salivary factor that has been shown to be efficacious in limiting blood-feeding when the recombinant form is used as an antigen.
Publications
- Cupp, M. S., E. W. Cupp, N.Wisnewski, K. S. Brandt, G. M. Silver, D. Zhang and V. Panangala.. 2004. Evaluation of a recombinant salivary gland protein (thrombostasin) as a vaccine candidate to disrupt blood-feeding by horn flies. Vaccine 22: 2285-2297.
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Progress 06/01/04 to 12/31/04
Outputs
The full-length structure of a gene encoding a novel antihemostatic factor (HFX) found in horn fly saliva was described. The HFX gene had 444 base pairs (bp) encoding 148 amino acids. Recombinant HFX, produced in both E. coli and insect SF9 cells, was immunogenic in rabbits and cattle.
Impacts
This newly-described salivary factor could prove important in the development of a polyvalent anti-feeding vaccine directed against the horn fly. It differs in structure from thrombostasin, another salivary factor that has been shown to be efficacious in limiting blood-feeding when the recombinant form is used as an antigen.
Publications
- No publications reported this period
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