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United States Department of Agriculture

Agricultural Research Service

2007 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. BSL-3; Pending.

3.Progress Report
Field sites have been identified for collection of Lutzomyia apache in Colorado. Some adult female flies have been collected to establish a colony.

The possibility of mechanical transmission of VSV by C. sonorensis was examined by testing frass from infected insects for virus by immunoblotting, real time PCR and virus isolation. Preliminary results show that VSV antigen can be detected by immunoblotting and VSV mRNA can be detected by rt-PCR. Infectious virus has yet to be isolated.

Transovarial VSV transmission studies have begun using C. sonorensis. Preliminary results suggest that VSV can be vertically transmitted from infected adult females to their progeny. F1 eggs, larvae, and emerged adults have been confirmed as VSV positive by real time PCR, western blotting, and virus isolation.

ABADRL is working with the Wyoming State Game and Fish Department (WYSGFD) and the USDA-ARS virus and prion disease of livestock research unit at Ames, Iowa to study the possible roles of insects in mechanical transmission of Chronic Wasting Disease (CWD). Preliminary research at a WYSGFD outdoor facility identified several arthropod species feeding on CWD infected elk or elk by-products. Over 300 adult insects have been collected, identified, and are stored at -80oC for prion detection. A subset of blood fed stable flies was pulled for blood meal identification.

ABADRL is working with Colorado State University, Department of Microbiology, Immunology and Pathology to study the possible role biting flies play in transmitting CWD. Six cages containing 300-500 female C. sonorensis midges were allowed to feed on blood spiked with CWD positive brain material or unspiked blood. Two cages were frozen at each time point: immediately post feeding, three days post feeding, and seven days post feeding. Prion detection assays are being optimized for testing insects. Mouthparts, midguts and salivary glands will be tested for prion contamination, uptake, and dissemination respectively.

A newly established method of collecting secreted C. sonorensis saliva has been used to obtain quantities of clean saliva for analysis by two-dimensional electrophoresis. Thus far, approximately 20 protein spots have been analyzed by mass spectrophotometry.

Midguts have been dissected from C. sonorensis given water, sugar water, blood, or blue tongue virus spiked blood to look for differences in protein expression in the midgut over time.

Experiments were performed to determine bluetongue virus (BTV) persistence in colonized C. sonorensis. Assays developed can distinguish between competent, virus persistent, and virus RNA free specimens.

Blood meal analyses were performed on field collected mosquitoes in the Powder River Basin, WY and Medicine Lake, MT. Results indicate a wide range of hosts including humans, livestock, and wildlife.

Determinations of the presence of West Nile virus (WNV), BTV, and Q fever in field collected specimens were made and are continuing. WNV and BTV have been confirmed in some insect samples.

Colonization of Three Mosquito Species. ABADRL has been redirected to work on Rift Valley Fever virus. Because this virus is thought to be transmitted by mosquitoes in areas where it is endemic or epidemic, a critical need for pertinent colonized mosquito species has become apparent. The initial colonization of three mosquito species (Ae vexans, Ae dorsalis and Cx tarsalis) has been completed. Currently, these must be maintained by forced copulation while ongoing attempts are made to provide conditions conducive to mating in captivity. These insects can now be used for research on virus-vector-host interactions at the ABADRL, and for studies that will be done on Rift Valley Fever virus vector competence. This accomplishment addresses the National Program Component 3 (Biology, Physiology and Vector – Pathogen Interaction). It is consistent with the need to protect the health of US livestock and reduce economic impact, to maintain the competitiveness of export trade markets, and provide a technical basis for new control technologies and scientifically based animal disease regulations.

5.Significant Activities that Support Special Target Populations

6.Technology Transfer

Number of new CRADAs and MTAs2
Number of active CRADAs and MTAs5
Number of U.S. patents granted1
Number of web sites managed1
Number of non-peer reviewed presentations and proceedings8
Number of newspaper articles and other presentations for non-science audiences1

Review Publications
Hunt, G.J., Schmidtmann, E.T. 2005. Care, maintenance, and experimental infection of biting midges. The Biology of Disease Vector 2nd Edition.

Langner, K.F.A., K.E. Darpel, E. Denison, B.S. Drolet, W. Leibold, P.S. Mellor, P.P.C. Mertens, M. Nimtz, I. Greiser-Wilke. 2007. Collection and analysis of salivary proteins from the biting midge Culicoides nubeculosus (Diptera: Ceratopogonidae). Journal of Medical Entomology 44(2):238-248.

Schmidtmann, E.T. 2006. Testing the relationship between dissolved salts in aquatic habitats and immature populations of the culicoides variipennis complex (diptera:ceratopogonidae). Environmental Entomology 35(5):1154-1160.

Schmidtmann, E.T., J. Lloyd, R.J. Bobian, R. Kumar, J.W. Waggoner, W.J. Tabachnick,and D. Legg. 2001. Suppression of mosquito (Diptera: Culicidae) and black fly (Diptera: Simuliidae) blood feeding from hereford cattle and ponies treated with permethrin. Journal of Medical Entomology. 38(5):728-734.

Borowicz, V., Alessandro, R.T., Albrecht, U., Mayer, R.T. 2005. Effects of nutrient supply and below-ground herbivory by Diaprepes abbreviatus L. (Coleoptera: Curculionidae) on citrus growth and mineral content. Applied Soil Ecology. 28:113-124.

Williams, Martin R., Juan Garcia, Kristine E. Bennett, Bernarda Molina, Stephen E. Aspen, and Harry M. Savage. 2007. First Records for Culex (Melaniconion) limacifer Komp and Culex (Melaniconion) dunni Dyar from Guatemala. 2007. JAMCA. 23(1):78-79.

O'Toole, D., Perez De Leon, A.A., Hearne, C., Mcholland, L.E., Yun, L., Tabachnick, W. 2003. Papular Dermatitis Induced in Guinea Pig by Biting Midge Culicoides Sonorensis (Diptera: Ceratopogonidaie). Journal of Veterinary Diagnostic Investigation. 15:67-71.

Mullens, B., Gerry, A., Lysyk, T., Schmidtmann, E.T. 2004. Environmental effects on vector competence and virogenesis of bluetongue virus in Culicoides: interpreting laboratory data in a field context. Veterinaria Italiana 40(3):160-166.

Last Modified: 9/10/2014
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