2008 Annual Report 1a.Objectives (from AD-416) The goal of research is to protect U.S. livestock from vector-borne pathogens through knowledge of species vector competence, defining the influence of environmental factors on vector competence and distribution, and understanding insect susceptibility to pathogen infection at cellular and molecular levels. Within this objective, research will:. 1)clarify and define the role of insects as vectors and reservoirs of pathogens that adversely affect livestock, humans, and wildlife;. 2)determine the effects of environmental factors on transmission of pathogens by biting midges and mosquitoes; and. 3)identify and characterize insect genomic and proteomic determinants that affect infection, replication, and transmission of arboviruses of concern to the U.S. livestock industry. 1b.Approach (from AD-416) Ecological data will be developed for criteria used to incriminate insect species that serve as vectors of obscure or little studied pathogens in nature. Emphasis will be placed on clarifying the role of sand flies, grasshoppers, and Culicoides as vectors and reservoirs of vesicular stomatitis viruses, as well as the role of both blood feeding and non-blood feeding insects in the natural transmission of prions. The role of environmental factors as determinants of insect vector distribution and susceptibility to arboviruses will be developed using geographic information system approaches to estimate vector distribution and pathogen susceptibility, thus enabling prediction of livestock risk for pathogen exposure. The midgut microbial flora of biting midges will be assessed using non-culture based molecular tests, such as T-RFLP, quantitative PCR or macro-arrays, and correlated with insect susceptibility to arbovirus infection. Arbovirus susceptibility and infection pathways in insect vectors will be investigated by identifying salivary gland and midgut proteins that will be analyzed by mass spectromety and compared with available protein databases and compared between blood-fed and non-blood fed individuals. RNA interference (RNAi) will be used to down regulate genes and thus identify the role of gene products in the susceptibility of insects to arbovirus infection. 3.Progress Report The following work supports NP104 Action Plan Component 1: Ecology and Epidemiology. Transovarial transmission studies of VSV in C. sonorensis were completed, and a manuscript is in preparation. VSV was transmitted from infected females to offspring as detected by real time PCR, western blot and virus isolation in eggs, larvae, and adult progeny. Frass from infected C. sonorensis was tested for VSV by immunoblotting, real time PCR and virus isolation to look for mechanical transmission. Results indicate that VSV can be detected by immunoblotting and real time PCR. Infectious virus was not isolated. Experiments continue to look for persistence of BTV in a C. sonorensis colony. Partial sequence of the L3 gene was identified but neither full length L3 nor other viral genes have been recovered. L. apache was found in WY and CO. Several hundred flies were collected and tested for VSV, Phleboviruses and other pathogens. No viruses were detected. An egg derived cell line was initiated. Laboratory feedings indicate that L. apache feeds on reptiles and not mammals. A near infrared spectroscopy tool for age grading C. sonorensis was developed in collaboration with the USDA-ARS agricultural engineering unit in Manhattan, KS. C. sonorensis from Montana were found to be susceptible to BTV and EHDV. C. sonorensis were found to be susceptible to Weldona virus. Cimicid bugs were found to be susceptible to VSV and could vector VSV among bats. No evidence of prions was detected for C. sonorensis tested immediately after feeding on CWD-spiked blood, and no insect homolog of the PRNP exists to enable prion conversion/amplification. Detection of Scrapie protein in laboratory exposed stable flies was successful. The departure of a key collaborator has stopped further work. The following work supports NP104 Action Plan Component 2: Detection and Surveillance Technology. A new infrared PCR tool was developed for EHDV and applied to field caught insects from MT and WY. The following work supports NP104 Action Plan Component 3: Biology and Physiology. The effects of adult sugar source, host blood chemicals, and larval temperature were studied in relation to BTV and EHDV. Salt balancing organs of C. sonorensis were identified and could be used to study environmental effects of ions on larvae. The effect of blood-borne bacterial pathogens of sheep on BTV susceptibility in C. sonorensis were studied using Bartonella spp. spiked blood meals, and had no effect. The following work supports NP104 Action Plan Component 4: Control Technology Progress was made in testing and validating formulations of long lasting pyrethroid pesticides for control of BTV in sheep. Permits for their use in WY and MT were approved. Two of three new treatments tested to control sheep keds showed significant promise. Methoprense S and Spinosad toxicity to larval C. sonorensis were demonstrated. 4.Accomplishments 1. Blood feeding of Vesicular Stomatitis Virus (VSV) infected Culicoides sonorensis. Numerous studies have evaluated the ability of C. sonorensis to transmit viruses, but none have looked at the potential for infected midges to exhibit behavioral modifications that affect blood feeding success and ultimately virus transmission. A study was initiated, completed, and has been accepted for publication on the effects of infection with VSV on Culicoides sonorensis blood feeding behavior. Results show that infection with VSV causes a transient decrease in blood feeding success that seems to correspond to peak virus titer in the insect. A delay in blood feeding could have significant impact on virus transmission and maintenance. This research provides further information toward a better understanding of the role of C. sonorensis in VSV transmission. 2. Determine potential Blue Tongue Virus (BTV-1) vectors in Louisiana. BTV-1 had not been reported in the USA prior to 2004 when it presented in a deer harvested in St. Mary Parish, LA. To determine potential vectors for autochthonous transmission, over 5000 biting midges from this region were collected in 2006 and 2007. Although no BTV-1 was isolated from collected samples, indigenous Culicoides species diversity was determined. Species of Culicoides identified include: C. arboricola, C. crepuscularis, C. debilipalpis, C. paraensis, C. hinmani, C. furens, C. stelifer, C. haematopotus, and C. biggutatus. Specimens of two other genera of ceratapogonids were caught in the traps: Forcipomyia and Atricopogon. The results of this study should lead to identification of the species of Culicoides that are competent vectors of BTV-1. The geographic range of the vector species will predict whether natural barriers to the spread of BTV-1 in the United States exist. This was the final component of Reimbursable Cooperative Agreement #58-5410-6-427 with LSU. 6.Technology Transfer Number of the New MTA (providing only) 2 Number of New Commercial Licenses Executed 11 Number of Web Sites Managed 3 Number of Non-Peer Reviewed Presentations and Proceedings 6 Review Publications Perez De Leon, A.A., Tabachnick, W.J. 2006. Transmission of Vesicular Stomatitis New Jersey Virus to Cattle by the Biting Midge Culicoides sonorensis (Diptera: Ceratopogonidae). Journal of Medical Entomology 43(2):323-329. Perez De Leon, A.A., O'Toole, D., Tabachnick, W.J. 2006. Infection of Guinea Pigs with Vesicular Stomatitis New Jersey Virus Transmitted by Culicoides sonorensis (Diptera: Ceratopogonidae). Journal of Medical Entomology 43(3):568-573. Bishop, J.V., Mejia, J.S., Perez De Leon, A.A., Tabachnick, W.J., Titus, R.G. 2006. Salivary gland extracts of Culicoides sonorensis inhibit murine lymphocyte proliferation and NO production by macrophages. American Journal of Tropical Medicine and Hygiene 75(3):532-536. Paddock, C.D., Fernandez, S., Echenique, G.A., Sumner, J.W., Reeves, W.K., Zaki, S.R., Remondegui, C.E. 2008. Rocky Mountain Spotted Fever in Argentina. American Society of Tropical Medicine and Hygiene. 78, 687-692. Reeves, W.K., Dillion, Jr, R.T., Dasch, G.A. 2008. Freshwater snails (Mollusca: Gastropoda) from the Commonwealth of Dominica with a discussion of their roles in the transmission of parasites. American Malacological Society Bulletin. Vol 24: p. 59-63. O'Toole, D., Perez De Leon, A.A., Hearne, C., Mcholland, L.E., Yun, L., Tabachnick, W.J. 2003. Papular dermatitis induced in guinea pigs by the biting midge Culicoides sonorensis (Diptera: Ceratopogonidae). Journal of Veterinary Diagnostic Investigation. 15:67-71. Nelder, M.P., Lloyd, J.E., Loftis, A.D., Reeves, W.K. 2008. Molecular detection of Coxiella burnetii in wild caught filth flies. Emerging Infectious Diseases. Vol 14:1002-1004 Adler, P.H., Roach, D., Reeves, W.K., Flanagan, J.P., Morrow, M.E. 2008. Attacks on the endangered Attwarer's Prairie-Chicken (Tympanuchus cupido attwateri) by black flies infected with an avian blood parasite. Journal of Vector Ecology. Vol. 32, No. 2, p. 309-312. Reeves, W.K., Loftis, A.D., Nicholson, W.L., Czarkowski, A.G. 2008. Acute Illness Associated with Ehrlichia SP P-MTN from Atlanta GA, USA: A Case Report. Journal of Medical Case Reports. Vol. 2:139. Reeves, W.K., Murray, K.O., Meyer, T.E., Bull, L.M., Pascua, R.F., Holmes, K.C., Loftis, A.D. 2008. Serological evidence of typhus group Rickettsia in a homeless population: Houston, Texas. Journal of Vector Ecology.33(1):206-208 Reeves, W.K. 2008. Molecular Evidence for a Novel Coxiella from Argas monolakensis (Acari: Argasidae) from Mono Lake, California, USA. Experimental and Applied Acarology. Vol 44:57-60 Rochlin, I., Santoriello, M.P., Mayer, R.T., Campbell, S.R. 2007. Improved high-throughput method for molecular identification of Culex mosquitoes. Journal of the Mosquito Control Association. 23 (4):488-491,2007 Langner, K., Darpel, K.E., Drolet, B.S., Fischer, A., Hampel, S., Heselhaus, J.E., Mellor, P.S., Mertens, P., Leibold, W. 2008. Comparison of cellular and humoral immunoassays for the assessment of summer eczema in horses. Veterinary Immunology and Immunopathology. 122: 126-137.