Progress 06/01/03 to 05/31/06
Outputs
The horn fly, Hematobia irritans (L.), is an important pest of livestock because adults are aggressive blood-feeders. Remarkably, even though horn fly adults feed recurrently on their hosts as ectoparasites, these flies lack the ADP-responsive antiplatelet aggregation and vasodilatory antihemostatic systems described for other blood-feeding Diptera. Horn fly salivary gland extracts do interfere with the normal coagulation process as demonstrated by the recalcification time assay. Using this as a baseline, the effects of saliva on recalcification time, activated partial thromboplastin time, prothrombin time, and thrombin time were measured to determine which arm(s) of the coagulation cascade might be impacted. Factor-deficient plasma assays also were used to measure possible perturbations in clotting. Gland-free saliva delayed the recalcification time as well as the activated partial thromboplastin time, prothrombin time, and thrombin time. Saliva also further delayed
clotting times of plasmas deficient in factor V, factor VIII, and factor XIII, indicating that other factors in the coagulation cascade were inhibited. Although horn fly saliva did not alter the ability of deficient plasma reconstituted with factor X to clot, it did inhibit deficient plasma reconstituted with factor II (thrombin). Antithrombin activity in saliva was confirmed by its ability to interfere with thrombin hydrolysis of fibrinogen, its normal substrate, and by its inhibition of thrombin action on a chromagenic substrate that mimics the hydrolytic site of fibrinogen. Horn fly saliva contains a factor that targets thrombin, a key component in the coagulation cascade. While the biochemical mechanisms of inhibition may vary, this antihemostatic characteristic is shared with other zoophilic Diptera such that feed on ungulates. Thrombostasin (TS) is a thrombin inhibitor found in the salivary glands of horn flies. It is produced as an inactive form with a 76-amino acid propeptide
in the N-terminus preceding the mature TS. A minimal recognition sequence by subtilisin-like proprotein convertases, Arg-Xaa-Xaa-Arg, is localized C-terminal to the propeptide. This study demonstrated that a gene cloned from the salivary glands of the horn fly encodes a new convertase, subsequently named horn fly proprotein convertase (HFPC), and that the recombinant HFPC expressed in insect HighFive cell culture specifically cleaves recombinant pro-thrombostasin, produced in E. coli, at the expected site. The relative cleavage efficiency of rHFPC was compared with that of recombinant human furin, a commercially available proprotein convertase. The result indicated that this newly identified proprotein convertase is of importance for the proteolytic maturation of thrombostasin, a protein secreted in horn fly saliva and used by the insect to counteract its host's haemostatic response. The outputs of this project have been disseminated through oral and poster presentations at
professional meetings, through classroom teaching efforts, and through refereed journal publications. In addition, the outputs of this project have been discussed with various industry scientists and with the public.
Impacts
This research addresses an important national priority and a successful outcome will significantly improve beef cattle and milk production in Alabama and the region by significantly decreasing the effects of horn fly ectoparasitism. Findings from this research could lead to an effective vaccine to prevent blood-feeding by the most important ectoparasite of cattle in North America. These findings are currently being evaluated by industry and could lead to a marketable product that will help cattlemen and dairymen.
Publications
- Procunier, W. Zhang, D. Cupp, M.S. Miller, M. Cupp, E.W. 2005. Chromosomal localization of two antihemostatic salivary factors in Simulium vittatum (Diptera: Simuliidae). Journal of medical entomology. 42(5): 805-811.
- Zhang, D. Cupp, M.S. Cupp, E.W. 2004. Processing of pro-thrombostasin by a recombinant subtilisin-like proprotein convertase derived from the salivary glands of horn flies (Haematobia irritans). Insect biochemistry and molecular biology. 34(12): 1289-1295.
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Progress 01/01/05 to 12/31/05
Outputs
Sufficient quantities of the two salivary factors (thrombostasin, hematollogen) were produced in the laboratory for large-scale vaccine trials which were initiated in March, 2004, and March, 2005. Volumes of blood consumed in a 20 min feeding time were significantly less for horn flies feeding on calves immunized with recombinant salivary antigens when compared to flies feeding on OVA-immunized control calves (ANOVA, n=855; p= 0.001).Mortality of flies following 40 hrs of continuous blood feeding on immunized calves appeared to be greater when the host was a calf immunized with salivary proteins compared to OVA controls. When differences in mortality were compared using data from only groups where values were within a similar range, the increase in mortality of flies feeding on saliva-vaccinated cattle was further supported (Kruskal-Wallis Test Statistic = 10.868,n = 118; p = 0.012). These findings indicate that a vaccine directed against the horn fly's
anti-hemostatic salivary secretions could be efficacious and therefore offers a completely new approach to the control of this important ectoparasite.
Impacts
Findings from this research could lead to an effective vaccine to prevent blood-feeding by the most important ectoparasite of cattle in North America. These findings are currently being evaluated by industry and could lead to a marketable product that will help cattlemen and dairymen.
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.
- Zhang, D., M. Cupp and E. Cupp. 2004. Processing of pro-thrombostasin by a recombinant subtilisin-like proprotein convertase derived from the salivary glands of horn flies (Haematobia irritans). Insect Biochem. Mol. Biol.34: 1289-1295.
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Progress 01/01/04 to 12/31/04
Outputs
The potential for controlling blood-feeding by the horn fly (Haematobia irritans) was tested by vaccination against thrombostasin (TS), an inhibitor of mammalian thrombin that is produced in the fly's salivary glands and released into the skin during blood-feeding. This inhibitor is produced as an inactive form which must be cleaved by a subtilisin-like proprotein convertase to be activated. The increase in blood meal size that normally occurs when flies feed on sensitized non-vaccinated hosts was blocked and egg development in female flies was delayed when horn flies fed on cattle immunized with recombinant TS.
Impacts
This research addresses an important national priority and a successful outcome will significantly improve beef cattle and milk production in Alabama and the region by significantly decreasing the effects of horn fly ectoparasitism.
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.
- Zhang, D., M. S. Cupp and E. W. Cupp. 2004. Processing of pro-thrombostasin by a recombinant subtilisin-like proprotein convertase derived from the salivary glands of horn flies (Haematobia irritans). Insect Biochem. Mol. Biol.34:1289-1295.
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Progress 01/01/03 to 12/31/03
Outputs
A major isoform of thrombostasin(TS9)has been expressed in sufficient quantities in E. coli to evaluate its importance as a vaccine candidate. This is the second isoform of thrombostasin to be expressed as a recombinant protein. Initial studies in rabbits indicate that the rTS9 is immunogenic, with an antibody titer of 1:256,000 recorded after 1 prime and two boosts. With the previous success expressing the TB8 TS isoform, this finding indicates that a multi-valent vaccine consisting of several recombinant TS isoforms can be made. Immunogenic trials with cattle will begin in early summer, 2004.
Impacts
This research addresses an important national priority and a successful outcome will significantly improve beef cattle and milk production in Alabama and the region by significantly decreasing the effects of horn fly ectoparasitism.
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 (in press). 1
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