Performing Department
Veterinary Sciences
Non Technical Summary
Bluetongue virus (BTV, genus Orbivirus, family Reoviridae) is an insect-transmitted virus with recently expanded geographic range worldwide, including severe outbreaks in northern Europe (previously free of the virus), and 10 new serotypes identified in the United States since 1999, changes largely attributed to a warming climate. BTV causes highly fatal disease in deer and pronghorn, and variable disease in livestock. Outbreaks in sheep flocks result in disease, production losses and mortality, as well as the economic impact resulting from quarantine of the farm. Cattle may be infected but rarely become sick, but producers suffer significant economic impacts from trade restrictions when animals test positive for BTV antibodies. Eastern Wyoming has high incidence of BTV infection with >88% of cattle test antibody positive, preventing farmers in this region from the ability to sell seed stock internationally. Currently, the annual BTV infection rate, time-of-onset of infections in the summer, and effect of meteorological variables are largely unknown. In addition, the extent and duration of protection afforded to calves and lambs from maternal antibodies is not known. We hypothesize that time of onset and intensity of natural BTV transmission will be correlated with climate conditions and maternal antibody levels.Over three summers, insect vector abundance will be assessed and spring born calves positive or negative for maternal antibodies will be sampled and tested for when they are first infected and the level of virus in their system. We will draw blood samples from calves twice a month throughout the summer as well as recording on-site daily weather conditions and collecting the insect vector. We will analyze the findings to determine correlations between BTV transmission and weather patterns. These findings will greatly advance our understanding of the meteorological conditions impacting risk for BTV infections and help in making predictions of changes likely to occur in a warming climate. Understanding the impact of climate variables on the transmission of BTV in range managed beef cattle will help to better predict outbreaks so livestock owners can plan preventative control strategies such as pursuing vaccines, pest control, and protective animal housing. We will also compare infection onset and intensity in calves born to antibody negative and antibody positive mothers. This will allow us to assess the duration and amount of protection provided to calves born to mothers previously vaccinated or naturally infected with BTV. This will help producers in making the difficult cost/benefit decisions that are a challenge to running a profitable business. This information will be significant in deciding to pursue dam and calf vaccination strategies based on the current antibody status of the herd.
Animal Health Component
100%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Major Goals and Objectives.Major Goals. Our goals are to better understand the dynamics of natural BTV transmission, how maternal antibody protection impacts this process, and identify correlated meteorological conditions. This information is needed to understand the current epidemiology of BT disease, better predict outbreaks, identify likely impacts of a warmer climate, and will help livestock owners make informed decisions on vaccination or other potential prevention strategies. Another valuable product of this research will be to isolate and identify the current BTV serotypes circulating in eastern Wyoming, information needed to recognize when new strains move into the state.Objectives.Identify climate variables correlated with the onset and intensity of BTV infection in beef cattle managed under range conditions, as well as vector prevalence.Determine the effect of maternal antibodies on time of onset of seroconversion to BTV in the first summer of spring born calves.Characterize the strains of BTV circulating at the study site during each of the three seasons of this study.
Project Methods
Study design: The study design is a prospective study over the course of three years, following the spring-born calves throughout their first summer to detect the time of onset and concentration of virus and virus specific antibodies.Study site, animal selection, and seasonal sample collection: The site of the study will be the University of Wyoming Sustainable Agriculture Research and Extension Center (SAREC) in Lingle, Wyoming. We will use 40 beef cow/calf pairs (40 cows and their calves), 20 each dams antibody positive and antibody negative, housed on common, nonirrigated rangeland pasture in Goshen County, a region with high seroprevalence (>88%) in cattle and sheep, and the SAREC cow herd (N=50) tested in 2015 had a seroprevalence of 100%. This gives us confidence that this study site is ideal for natural exposure.Cows and calves will be kept on grass pasture typical of range fed cattle in Wyoming. Blood samples will be collected at time of branding in April, prior to the expected transmission season, to identify initial antibody status of the cows, and to verify that calves of positive mothers have acquired maternal antibodies. Calves will then be sampled every other week from June to September with a final sample collected at the end of October or after the first hard frost (<-7 C). BTV infections are distinctly seasonal in regions with winters with freezing temperatures. The BTV insect vector (Culicoides sonorensis) has been shown to have optimal temperature range for development 25-35C with a potential range for development and survival from 9 C to >35 C (upper range not tested). In Lingle, these temperatures are not reached as an average daily temperature until May (13 C). Thus, by first testing cows in April we will only detect antibodies elicited by an infection in a prior season.Animal Use Protocols have already been approved for this project by the University of Wyoming Institutional Care and Use Committee. Cows will have blood collected from the ventral tail vein using a 1" 18 G needle and calves from the jugular vein with a 1-1.5' 20 G needle. A serum tube will be collected for antibody assays, and a tube with EDTA anticoagulant will be used for RNA extraction and RT-PCR.Antibody detection assays.Serum will be separated and stored at -80 C prior to testing for BTV specific antibodies by competitive enzyme linked immunosorbent assay (cELISA), and assay that does not determine serotype. Samples testing positive by cELISA will be further tested for virus serotype and antibody concentration (titer) by standard microwell neutralizing assays, using duplicate wells of 4-fold serial dilutions from 1:20 to 1:2560.Virus detection assays.RNA extraction and Real-time RT-PCR. RNA will be extracted from anticoagulated (EDTA) whole blood using the Ambion MagMax RNA extraction kit following manufacturer's instructions, then tested for virus using a real-time RT-PCR assay that detects and distinguishes BTV and EHDV. This assay has been validated in our laboratory and has an efficiency (based on serial 10x dilutions in triplicate: {10(-1/slope)}-1) of 97% for BTV and 98% for EHDV. An internal control is added to each sample prior to extraction to monitor assay performance and determine the success of the RNA extraction or the presence of inhibitory factors. Positive and negative controls are included each time the assay is run.Virus Isolation. Samples testing positive by RT-PCR will be tested for infectious virus by virus isolation assay using cattle pulmonary arterial endothelial cells (CPAE) which are susceptible to infection with BTV and EHDV. Whole blood samples are hemolyzed, RBC membranes pelleted, washed with 10 x volume PBS, pelleted again and resuspended 1:10 (v/v) in tissue culture media (MEM, P/S, 4% FBS) and applied to confluent CPAE cells in duplicate wells of a 24-well plates drained of media. After incubation at 37 C for 1 hour, media is replaced and the cultures are observed daily for the development of cytopathic effect (CPE). Wells negative for CPE after 7 days are frozen, thawed, and the media applied to new cells for a single blind passage.Virus serotype and genotype determination. Serotype of the infecting virus will be tested by ability of antibody positive serum samples to neutralize the two serotypes previously identified in Wyoming, BTV-11 and BTV-17. Samples positive by group specific antibody test (cELISA) but negative for BTV-11 and 17 will be forwarded to the National Veterinary Service Laboratory for typing to other serotypes in the U.S. An isolate of each serotype identified each year will be grown and archived for complete genome sequencing in future studies.Vector trapping, identification, and virus detection.We will collect the insect vector and determine meteorological conditions at which the insects are most active. We will trap, identify, and count Culicoides two days a week throughout the summer. This will allow us to compare relative trap counts with temperature, humidity and wind conditions on that day and the preceding two days, and other factors can be considered as well. The relative counts are a measure of vector numbers as well as activity, since even if the population numbers are high, if weather conditions are unfavorable (cold or windy), the bugs are likely to be cloistered at the ground. Hence, our counts will be a relative measure of the number of Culicoides active on that day.Modified CDC mini down-draft light traps with black light will be placed in two locations, 15 m apart near a common water source used by the cattle in the study. Collected insects are first separated for Culicoides spp. and other insects (bycatches) under a stereomicroscope. Afterwards the Culicoides spp. complexes are determined by the characteristic pattern and coloration of the wings with the assistance of Dr. Will Reeves, entomologist, epidemiology consult services, USAF School of Aerospace Medicine. Pools of approximate 100 bugs will be homogenized in 1 ml of tissue culture media (MEM, P/S, 4% FBS) using a Tissue Lyzer, then processed for RNA extraction using Ambion MagMax RNA extraction kit. The remnant homogenates will be frozen at -80 C for virus isolation attempt of samples testing positive by RT-PCR.Statistical analysis.Sample size calculation. The inference for means: comparing two independent samples, was used to estimate the number of animals needed to show significance at alpha=0.05, and power 0.80 (Bernard Rosner's Fundamentals of Biostatistics). We assumed that antibody negative calves would have onset of infection >/= 4 weeks before the antibody positive calves, with a standard deviation of 3 weeks. Using a two sided test we estimate 9 animals per group will provide adequate power. For the summer of 2015, ten antibody negative calves and 20 maternal antibody positive calves were under study. For the summers of year 2 and 3, 20 calves will be in each group increasing the power of our analysis to 0.9 with a standard deviation of 4 weeks (estimated sample size 22).Climate variables (initially temperature, wind, humidity and rainfall) will be tested by using univariate logistic regression for correlation with presence of infection in the animals, and with relative numbers of vector trapped. The variables with a univariate p value of < 0.2 will then be fit to a Generalized Additive Model to identify non-linear relationships between different values of an explanatory variable and the outcome. A stepwise exclusion will be applied to remove the least significant variable with reanalysis applied until only variables with p<0.1 remain. Comparison of time of onset of infection and level of viremia between calves positive or negative for maternal antibodies, will be tested using the student's t-test for independent samples. A Cox Proportional Hazards model will also be fit to assess variables associated with time of onset of infection.