2006 Annual Report
48 month Milestone(s) Objective 1 1a Second season field sampling for the sand fly L. apache in southern New Mexico will be conducted. 1b No milestone. 1c The rate of vertical transmission of VSV (in Culicoides sonorensis) as determined by virus isolation in eggs of infected adults will be determined. 1d No milestone. 1e Determination of vertical transmission of prions in insects. Objective 2 The effects of selected environmental factors on infection of BTV in C. sonorensis will be conducted. Objective 3 3a Immunomodulatory salivary proteins will be cloned and expressed. Expressed immunomodulatory salivary proteins will be examined for their effects on virus infection. 3b Continued genetic studies of suspected vector competence genes to exotic species of Culicoides will identify species-specific genetic markers and potential vector competence genetic markers.
60 month Milestone(s) Objective 1 1a. Experimental transmission studies with L. apache will be carried out. 1b. Any virus isolated from grasshoppers will be partly sequenced and compared to known livestock isolates. 1c. The progression of VSV-GFP in developmental stages of Culicoides will be determined. 1d. No milestone. 1e. No milestone. Objective 2 GIS predictions of vector competence spatial relationships will be developed for C. sonorensis and Culex tarsalis in the Rocky Mountain region. Objective 3 3a No milestone. 3b Application of RNAi and other novel methods developed over the course of these studies for controlling insect vector infection will be examined and results published.
Mosquito repellent study. The results of a large-scale field trial in which cattle were treated with a commercially-available, over-the-counter insecticide/repellent product, show suppression of mosquito feeding by ca. 90% over a six week period. This research establishes a practical method that can be useful in limiting the spread of mosquito-transmitted pathogens by protecting naive animals, reducing infection of mosquitoes that feed from an infected animal, as well as providing immediate animal protection in lieu of vaccination or the development of protective antibodies.
The effects of temperature on the vector competence of the biting midge, Culicoides sonorensis, for bluetongue virus was determined for insects in Montana and Alberta Canada. The data indicates that the populations collected in Alberta and northern Montana are refractory (non-susceptible) to infection with bluetongue virus and supports the reduced testing of Montana cattle for bluetongue virus before export to Alberta feedlots.
During the first phase of this project ARS worked with the CSU Arthropod-borne Infectious Diseases Laboratory from May to August 2005 to learn the molecular tools for gene silencing. Two potential targets were identified and one was successfully cloned into a RNA transcription vector that was used to produce double-stranded (ds) RNA by in vitro transcription. The dsRNA produced was injected into Aedes aegypti to produce small inhibitory RNA (siRNA). The ability of this mosquito RNA silencing system to affect the replication of a Malaysian Sindbis strain MRE16 that replicates in Aedes aegypti midgut cells was evaluated. Preliminary results suggest that the target successfully reduced virus dissemination from the midgut. This technology has been transferred to ABADRL and studies are being conducted to confirm the findings. Eventually, the technology will be adapted for use in Culicoides. This project has been put on temporary hold so that emphasis can be put on completing other ongoing research projects prior to when the lab's redirection becomes effective.
5410-32000-014-11S. This report serves to document research conducted under Specific Cooperative Agreement # 58-5410-6-083F between ARS and Kenya Medical Research Institute (KEMRI) entitled "Genetic Studies of Rift Valley Fever Virus Vectors in Kenya". Specifically the aim of this study was to look at Aedes mcintoshi and Aedes circumluteolus gene flow in Kenya. The introduction of West Nile Virus into North America has shown that foreign disease agents clearly pose a threat to human health, but can also have important adverse effects on agriculture. Rift Valley fever virus (RVFV) is the focus of a relatively recent redirection in the research mission at the ABADRL lab in Laramie, WY. RVFV has not yet been identified in the United States, but the potential impact that its introduction would have justifies efforts to better understand the disease cycle in its entirety. Research must be done in areas where the virus is endemic and/or epidemic to fully accomplish this goal.
This project piggybacks existing research aims of colleagues in Kenya. Kenya tends to have epidemic outbreaks of RVF every 5-7 years, but little has been done to understand what happens to the virus/vectors/hosts during the inter-epidemic periods. Most studies focus on RVF epidemiology, primarily as it pertains to the vertebrate hosts, attributes of the virus itself, and transovarial transmission of the virus in its various invertebrate vectors. This project's aim is to study the genetics of known RVFV vectors in Kenya. Rates of gene flow among mosquito populations, looking at two or three vector species will be determined. Collection sites include locations already selected for use in epidemiological studies due to their proximity to past RVF, and will include locations on either side of the Great Rift Valley to determine whether this poses a geographic barrier to gene flow.
In fiscal year 2006, mosquitoes were collected as adults, larvae, and eggs. Adults were collected using backpack aspirators and identified to species. Larvae were brought to the lab, raised to adults (some raised individually as voucher specimen), identified to species and processed as adult collections. Eggs were brought back to the lab, hatched and processed as larvae. For species previously implicated in transovarial transmission (Aedes circumluteolus and Aedes mcintoshi), head and thoraces were placed in 100% ethanol for use in the gene flow study and abdomen were placed frozen at -80 for virus detection using RT-PCR. Mosquitoes were identified to species and prepared for DNA or RNA extraction for genetic analysis of mosquitoes and determination of the presence of RVFV.
5410-32000-014-03N. This report serves to document research conducted under Non-Funded Cooperative Agreement #58-5410-4-106N, "Radioactive Materials License", between ARS and the University of Wyoming. The University of Wyoming oversaw the ABADRL radioactive materials safety program and held the NRC license utilized by ABADRL staff. ABADRL staff participated in the training and advising of University of Wyoming's students as adjunct appointees.
5410-32000-014-01R. This report serves to document research conducted under Reimbursable Agreement #60-0000-5-2004, "Deployed Warfighter Protection Research Program", between the ARS and the U.S. Department of Defense. Construction for a new insectary that was partially funded from DOD funds awarded in FY2004 and FY2005 was completed in July 2006. Environmental chambers are being made operational; when this is completed and the chambers are checked out, mosquito colonies will be established to investigate insecticide effectiveness. No funding was awarded in FY 2006.
5410-32000-014-04N. This report serves to document research conducted under Non-Funded Cooperative Agreement #58-5410-4-104N, "Promote the Efficient and Safe Diagnosis and Surveillance of Emerging Arthropod-Borne Infections Diseases in Wyoming", between ARS and the Wyoming Department of Health, Cheyenne, WY. Insect vectors were trapped at designated areas in Wyoming and then sampled for West Nile virus via polymerase chain reaction techniques. This reporting period 9929 mosquitoes and midges were collected and of these 4682 were Culex tarsalis of which ca. 0.08% were infected with West Nile virus. West Nile was detected in 0.04% of all collected vectors (midges and mosquitoes). Mosquito and biting midge populations were sampled across Wyoming and tested for infection with West Nile virus (WNV) to determine virus infection rates and the host source of blood meals. In 2004 about 0.02% of mosquitoes (Culex tarsalis, C. pipiens, Aedes vexans, Psorophora sp., Culiseta inornata, Ochlerotatus dorsalis, O. melanimon, O. campestris, O. trivittatus) were positive for WNV, including approximately 0.1% infection rate in C. tarsalis. In 2005, the infection rate in all mosquito species surveyed was 0.04%, and 0.08% in C. tarsalis. Mosquito blood meal analyses revealed that C. tarsalis primarily feeds on mammalian species (deer, cattle, pronghorn, human, rabbit, moose, and vole), but also feeds on avian species (sparrow, pheasant, duck, hawk, oriole, snipe, owl and sage grouse). A. vexans showed a greater preference to mammals (host meals include: deer, pheasant moose, cow, pronghorn, human, vole and sparrow). A. dorsalis host blood meals (cow, pronghorn, deer, human, rabbit, and moose) reflect only mammalian species. In a similar study performed in Medicine Lake, MT (2005) vectors include A. vexans, Culiseta inornata, and C. tarsalis. The hosts in this area include deer, cow, human, dove, sharp-tailed grouse, pheasant, robin, moose, owl, pelican and turkey.
ABADRL scientists developed a high throughput IR dye diagnostic method for detection of bluetongue virus (BTV) in Culicoides that can be performed in less than 6 h (from insect to result). The IR-reverse transcriptase PCR (IR-RT-PCR) assay has a sensitivity of 0.5 pfu from an extraction of 10 pfu's. All 24 BTV serotypes can be detected. Epizootic hemorrhagic disease virus serotypes are apparently not detected by this method. The manuscript is written and is under review. 220 pools of Culicoides collected in Louisiana in 2003 and 2004, representing 46,496 midges were tested for the presence of BTV RNA by IR-RT-PCR. None were found positive.
5410-32000-014-06N. This report serves to document research conducted under Non-Funded Cooperative Agreement #58-5410-4-111N, "Detection of Arthropod-Borne Diseases and Protection of Livestock", between ARS and the Wyoming Department of Agriculture, Cheyenne, WY. Mosquitoes were trapped in the Powder River Basin of Wyoming and those who had visible blood meals were used to determine what the host source of the blood meal was to assist with identification of various sources of West Nile virus in the environment. Blood meal hosts were identified through amplification of the cytochrome b gene using PCR analyses. Assays were largely of three mosquito species. Culex tarsalis (72 individuals) had the widest host range including: deer, cow, pronghorn antelope, human, rabbit, sparrow, moose, pheasant, duck, hawk, oriole, snipe, vole, owl, sage grouse. Aedes vexans (93 individuals) had the next widest host range including: deer, pheasant, moose, cow, pronghorn antelope, human, vole, and sparrow. Aedes dorsalis (65 individuals) had the smallest host range including: cow, pronghorn antelope, deer, human, rabbit, moose.
5410-32000-014-07N. This report serves to document research conducted under Non-Funded Cooperative Agreement #58-5410-4-125N, "Research on Arthropod-Borne Diseases of Livestock and Wildlife and Their Vectors", between ARS and the University of Wyoming, College of Agriculture, Laramie, WY. Cooperative efforts with the University of Wyoming have slowed due to lack of adequate laboratory facilities. Facility renovation should be completed by the end of 2006.
5410-32000-014-02T. This report serves to document research conducted under Trust Agreement 58-5410-3-0019, "Effects of temperature on vector competence of the biting midge, Culicoides sonorensis, for bluetongue virus" with the ARS and Agriculture Canada. Because of concern over the potential risk for transmission of bluetongue virus in cattle exported from Montana to Alberta, field populations of the primary vector of bluetongue virus, C. sonorensis from northern Montana and Alberta are being challenged with bluetongue virus as a test of their oral susceptibility, a key component of vector competence. Flies challenged in 2004 and 2005 were held for 12 days at 22 degrees Centigrade to test the effects of higher temperature on virogenesis. Flies were tested for infectious virus by cell culture, and the presence of BTV RNA by PCR. 10% of the colony flies were positive by cell culture, and approximately 70% positive by PCR. None of the Alberta midges were positive by cell culture, yet 88% were positive by PCR. 1.4% of the Montana specimens were positive by cell culture, with 72% positive by PCR. PCR positives represent the presence of BTV RNA and do not necessarily reflect infectious virus. The data available to date suggest that the populations from which the test flies were collected in Alberta and northern Montana are refractory (non-susceptible) to infection with bluetongue virus. The 2004 and 2005 study confirmed previous data from 2003, and supports the reduced testing of Montana cattle for bluetongue virus before export to Alberta feedlots.
ABADRL field and laboratory research incriminated the biting midge, Culicoides sonorensis, as the primary, proven vector of bluetongue viruses in the U.S. Data developed by the ABADRL also established that the northeastern U.S. is bluetongue-free because it is outside the range of C. sonorensis. An ABADRL extensive investigation of the population genetics of the C. variipennis complex in North America resulted in a taxonomic revision of the species complex, in which the three subspecies were elevated to species status. In a follow-up study, it was demonstrated that levels and types of dissolved salts in larval habitats differ among aquatic habitats occupied by respective species. In a study to develop methods for assessing bluetongue disease-free regions in support of export sales for U.S. livestock, 74 farms and ranches across Nebraska, South and North Dakota were sampled for C. sonorensis, the primary vector of bluetongue viruses in the U.S. The results show that the presence of C. sonorensis populations is correlated with geographic regions where evaporation exceeds precipitation and soils are non-glaciated. This information will help to clarify the epidemiology of bluetongue in both enzootic and bluetongue-free regions, further illustrating that the presence of the vector C. sonorensis is a primary risk factor for the exposure of livestock to bluetongue viruses. These findings open the way for analysis of environmental factors that support the presence of C. sonorensis populations, as well as large scale mapping and prediction of C. sonorensis using computer-based GIS methods. The demonstration of bluetongue-free regions of the U.S. has resulted in a relaxation of export regulations, thus improving the competitiveness of U.S. livestock and livestock germplasm in world markets.
The role of C. sonorensis as a vector of vesicular stomatitis virus (VSV) has been examined in the laboratory using experimental procedures, such as immunohistochemical techniques, which show that VSV passes through the midgut barrier, and thus infected insects may be a competent biologic vector. VSV in the salivary glands further indicates that C. sonorensis is able to vector VSV during feeding. The presence of VSV in the eggs suggests that VSV may transfer vertically from females to their offspring. A protocol has been developed to isolate VSV RNA from paraffin embedded insects which can then be used for PCR analysis; this will enable confirmation of viral replication within insect tissues where virus is no longer viable. Nucleocapsid mRNA was successfully amplified from paraffin embedded Culicoides by RT-PCR. This confirmed VSV replication in the virus fed insects. An extensive time course in vivo analysis of VSV infection in the biting midge, Culicoides sonorensis showed that following ingestion, VSV replicated in salivary glands, ovarial tissues and was excreted. This evidence may suggest bite, vertical, and mechanical VSV transmission may occur in Culicoides sonorensis. Culicoides sonorensis was shown to be a biologically competent vector for vesicular stomatitis virus (VSV). Virus passed efficiently through the midgut and salivary gland barriers and replicated throughout the insect. Virus replication and production was detected in salivary glands from days 3-13 post feeding and in the eggs from days 5-13. This is the first report of the vector competence of Culicoides for VSV.
Recent ABADRL research identified a non-hematophagous insect (grasshopper) as a potential source of infection and maintenance reservoir of VSV. This study demonstrated that VSV replicates in grasshoppers and can spread between grasshoppers and cattle, which may lead to a better understanding of the epidemiology of sporadic outbreaks of VSV among western livestock. Analysis of the distribution of the sand fly, Lutzomyia apache, in the mid-Rio Grande River Valley, New Mexico, using geographic information system methods indicates a potential role for this sand fly in the epidemiology of VS viruses in the southwestern U.S. A series of samples of grasshoppers active on 10 livestock premises is Wyoming and Montana immediately following confirmation of VS-N J virus in horses was collected for laboratory assay of possible infection. VSV-NJ diagnostic base-pair sequences have been obtained, but these specimens have yet to be tested due to the unavailability of BSL-3 laboratory facilities required for work with this pathogen.
The ABADRL insect vector genomics program has identified genes for which transcription increases during virus infection. In addition, midgut and salivary gland cDNA libraries were prepared and sequenced. This information has been released to international databases and will allow the development of new hypotheses of vector capacity as well as models to predict and understand the epidemiology of these diseases. Research that utilizes the genetic markers and cloned genes produced by the ABADRL will increase our understanding at the molecular level of the vector competence of C. sonorensis for bluetongue and epizootic hemorrhagic disease viruses.
Genes that could be related to BTV and EHDV vector competence were identified and efforts were concentrated on evaluating the expression of these genes in two insect colonies with high and low susceptibility to infection. This information has provided a foundation for understanding the genetics of C. sonorensis vector competence. Gene sequence comparison studies were put on lower priority due to loss of critical personnel and redirection of research unit.
An EST database of genes expressed in the salivary glands and midguts of C. sonorensis has been published and is available through GenBank. Salivary glands and midguts express genes that could be related to vector competence and this information is being used in current research described in a related CRIS project. The results of this research provide a foundation for research to identify vector competence genes, as well as new strategies for targeted control of arboviral disease transmission systems. The ABADRL has received two requests for international collaborations as a result of this work.
An initial study comparing sequence variation and gene expression of suspected vector competence genes in Culicoides-competent and not-competent in BTV transmission was completed. Transcript levels of four genes that preliminary studies indicated may play a role in orbivirus susceptibility and resistance were in all life stages of the two colony populations that were evaluated. This study provided evidence for intersection between the highly regulated LAR and FZ2 integrin-mediated cell signaling pathways. Further characterization of the translation products and gene silencing experiments could better define these complex interactions.
A method for obtaining significant quantities of Culicoides sonorensis secreted saliva has been developed. This will allow salivary protein characterizations never possible before due to insufficient material for analysis.
Culicoides sonorensis linkage map. Culicoides sonorensis is the primary vector of bluetongue virus in the western USA. The availability of a linkage map for this species would provide an important tool for better understanding its genetic make up and potential targets for vector control. Additionally, it would provide the groundwork for mapping genes/gene groups that contribute to vector competence phenotypes.
To date, F1 intercross families have been created that represent all possible combinations of males and females from the three Culicoides sonorensis colonies that are currently on hand at the ABADRL. Several of these have been maintained through multiple generations as far as the F8 generation. Families have enough individuals to provide statistical significance to linkage analysis. They are ready to be used for linkage mapping. Primers specific to the Culicoides sonorensis genome have been designed from the EST database developed by ABADRL. Intercross families were created and are ready for use in SSCP analysis and linkage mapping, and primers have been designed for use in linkage mapping within fiscal year 2007.
Culicoides sonorensis mitochondrial genome sequencing. Because of difficulties in getting good PCR amplification and sequencing, primers have been redesigned to more closely resemble other dipteran mitochondrial sequences. This will allow for more rapid and reliable results.
Insectary building completion. Construction of a ~2000 ft2 insectary building has been completed. Before the end of calendar year 2006 all necessary installations will be completed (phone, DI water system, operational environmental growth chambers, etc.) with occupancy expected in FY2007. This will provide much needed space and controlled environments with which to raise consistently healthy midge and mosquito colony insects.
Collaboration with Kenya Medical Research Institute (KEMRI) was initiated and insects were collected for a gene flow study of RVFV vectors in Kenya. This will provide insect biology/physiology and ecology/epidemiology information for emerging/exotic vectors and vector-borne disease research.
In August 2006, Wyoming had the nation's index case for vesicular stomatitis virus, an unprecedented event in the reported history of the disease. Insect collections were made in Natrona County, Wyoming, on three premises where vesicular stomatitis virus was identified in horses to possibly obtain VSV over wintering information. Standard CDC light traps baited with dry ice were positioned in areas where infected horses had access, where uninfected horses were stabled, and where aquatic insect habitat (black flies and mosquitoes) was ideal for 3 nights. Grasshoppers were also collected at each site using sweep nets. These insects are currently being sorted and identified and will then be tested for the presence of VSV.
The salivary glands and midguts of C. sonorensis EST database has been made available to entomologist and vector biologist through the national genetic database, GenBank.
The pre-publication results of a large-scale field trial in which cattle were treated with a commercially-available, over-the-counter insecticide/repellent product, to suppress mosquito blood feeding and transmission of West Nile virus was presented at meetings attended by mosquito control program directors, veterinary entomologists, insecticide manufacturers, and Cooperative Extension Service personnel.
"Researcher sleuths Wyoming vectors for vesicular stomatitis" by Steven L. Miller reports Drolet’s work on VSV and Culicoides was published in "AgNews" Spring 2006 edition University of Wyoming, "College of Agriculture web news release" May 2006, and "Cooperative Extension Service Publication", May 2006. Information regarding Culicoides sonorensis biology was also provided to the National Geographic Channel, January 2006 and used for a film production of the biblical plagues.
Campbell, C.L., K. VanDyke, G.J. Letchworth, B.S. Drolet, and W.C. Wilson. Culicoides gene discovery project reveals putative pharmacological factors in midge saliva. Presentation, Entomological Society of America, Salt Lake City, UT.
White D.M., W.C. Wilson, C.D. Blair, and B.J. Beaty. Possible overwintering mechanism of bluetongue virus in the arthropod vector. Presentation, American Society for Virology Meeting. Davis, CA.
Campbell, C.L., and W.C. Wilson. Characterization of differentially expressed midge genes in orbivirus vector populations. Presentation, Am. Soc. Trop. Med. and Hygiene. Washington, D.C.
K. Bennett and E.T. Schmidtmann. Prospective ABADRL insect rearing on behalf of the Deployed War Fighter Protection Program, Presentation, College Station, TX
Drolet, B.S. Culicoides sonorensis as a potential biological vector for vesicular stomatitis virus. Presentation, American Society for Virology, Lexington KY. ARIS#136458
C.Y. Kato, A. Fabian, E.T. Schmidtmann, M. Doherty and G.D. Johnson. “Blood-feeding patterns of Wyoming mosquitoes: Cytochrome b analysis”. Presentation, Wyoming Mosquito Control Association Annual Meeting, Lander, WY.
L. Zou, S. N. Miller, and E. T. Schmidtmann. A GIS tool to estimate West Nile virus activity using a degree day model. Wyoming Mosquito Control Association Annual Meeting, Lander, WY.
E.T. Schmidtmann, J. Waggoner, W. Yarnell, and J.E. Lloyd. Biosecurity: protecting livestock from mosquito blood feeding and arbovirus transmission”. Presentation, Wyoming Mosquito Control Association Annual Meeting, Lander, WY.
Mayer, R.T. An update on the Arthropod-Borne Animal Diseases Research Laboratory, Laramie, Wyoming. Presentation, U.S. Animal Health Association meeting, San Diego, CA.
Campbell, C.L., K. VanDyke, D.L. Mummey, and W.C. Wilson. Advances in the biology of the biting midge, Culicoides sonorensis. Presentation, Keystone Symposia, Genetic Manipulation of Insects, Taos, NM.
E. T. Schmidtmann. The Culicoides variipennis complex and bluetongue virus in the United States. School of Veterinary Medicine, Department of Epidemiology and Biostatistics, University of Montevideo, Montevideo, Uruguay.
Muth, M.R., G.J. Hunt, and E.T. Schmidtmann. An alkaline hydrolysis tissue digestion system for a BSL-3 containment facility. In Anthology of Biosafety VI. Arthropod Borne Diseases, J.Y. Richmond (ed.), pp 99-111.
Hunt, G.J., and E.T. Schmidtmann. Care, maintenance, and experimental infection of biting midges. In The Biology of Disease Vector, 2nd Edition, J. Goddard (ed.), pp. 741-746.
E.T. Schmidtmann, J. Waggoner, W. Yarnell, and J.E. Lloyd. Protecting livestock from mosquito feeding and West Nile virus: a large-scale field trial testing the repellency of permethrin treatment. Proceedings 50th Livestock Insect Work Conference, Amarillo TX. p 93.
Nunamaker, R.A., Lockwood, J.A., Stith, C.E., Campbell, C.L., Drolet, B.S., Wilson, W.C., White, D.M., Letchworth III, G.J. 2003. Grasshoppers can serve as reservoirs and vectors of vesicular stomatitis virus. Journal of Medical Entomology. 40:957-963.
Mcholland, L. E., Mecham, J. O. 2003. Characterization of cell lines developed from field populations of Culicoides sonorensis (Diptera: Ceratopogonidae). Journal of Medical Entomology. 40 (3):348-351.