Estimating Regional Methane Emission Factors from Energy and Agricultural Sector Sources Using a Portable Measurement System: Case Study of the Denver-Julesburg Basin.

Methane (CH4), a powerful greenhouse gas (GHG), has been identified as a key target for emission reduction in the Paris agreement, but it is not currently clear where efforts should be focused to make the greatest impact. Currently, activity data and standard emission factors (EF) are used to generate GHG emission inventories. Many of the EFs are globally uniform and do not account for regional variability in industrial or agricultural practices and/or regulation. Regional EFs can be derived from top-down emissions measurements and used to make bespoke regional GHG emission inventories that account for geopolitical and social variability. However, most large-scale top-down approaches campaigns require significant investment. To address this, lower-cost driving surveys (DS) have been identified as a viable alternative to more established methods. DSs can take top-down measurements of many emission sources in a relatively short period of time, albeit with a higher uncertainty. To investigate the use of a portable measurement system, a 2260 km DS was conducted throughout the Denver-Julesburg Basin (DJB). The DJB covers an area of 8000 km2 north of Denver, CO and is densely populated with CH4 emission sources, including oil and gas (O and G) operations, agricultural operations (AGOs), lakes and reservoirs. During the DS, 157 individual CH4 emission sources were detected; 51%, 43% and 4% of sources were AGOs, O and G operations, and natural sources, respectively. Methane emissions from each source were quantified using downwind concentration and meteorological data and AGOs and O and G operations represented nearly all the CH4 emissions in the DJB, accounting for 54% and 37% of the total emission, respectively. Operations with similar emission sources were grouped together and average facility emission estimates were generated. For agricultural sources, emissions from feedlot cattle, dairy cows and sheep were estimated at 5, 31 and 1 g CH4 head-1 h-1, all of which agreed with published values taken from focused measurement campaigns. Similarly, for O and G average emissions for well pads, compressor stations and gas processing plants (0.5, 14 and 110 kg CH4 facility-1 h-1) were in reasonable agreement with emission estimates from intensive measurement campaigns. A comparison of our basin wide O and G emissions to measurements taken a decade ago show a decrease of a factor of three, which can feasibly be explained by changes to O and G regulation over the past 10 years, while emissions from AGOs have remained constant over the same time period. Our data suggest that DSs could be a low-cost alternative to traditional measurement campaigns and used to screen many emission sources within a region to derive representative regionally specific and time-sensitive EFs. The key benefit of the DS is that many regions can be screened and emission reduction targets identified where regional EFs are noticeably larger than the regional, national or global averages.

Modeling air emissions from complex facilities at detailed temporal and spatial resolution: The Methane Emission Estimation Tool (MEET).

Recent attention to methane emissions from oil and gas infrastructure has increased interest in comparing measurements with inventory emission estimates. While measurement methods typically estimate emissions over a few periods that are seconds to hours in length, current inventory methods typically produce long-term average emission estimates. This temporal mis-alignment complicates comparisons and leads to underestimates in the uncertainty of measurement methods. This study describes a new temporally and spatially resolved inventory emission model (MEET), and demonstrates the model by application to compressor station emissions - the key facility type in midstream natural gas operations The study looks at three common facility measurement methods: tracer flux methods for measuring station emissions, the use of ethane-methane ratios for source attribution of basin-scale estimates, and the behavior of continuous monitoring for leak detection at stations. Simulation results indicate that measurement methods likely underestimate uncertainties in emission estimates by failing to account for the variability in normal facility emissions and variations in ethane/methane ratios. A tracer-based measurement campaign could estimate emissions outside the 95% confidence interval of annual emissions 30% of the time, while ethane/methane ratios could be mis-estimated by as much as 50%. Use of MEET also highlights the need to improve data reporting from measurement campaigns to better capture the temporal and spatial variation in observed emissions.

Identical Viral Genetic Sequence Found in Black Flies (Simulium bivittatum) and the Equine Index Case of the 2006 U.S. Vesicular Stomatitis Outbreak.

In 2006, vesicular stomatitis New Jersey virus (VSNJV) caused outbreaks in Wyoming (WY) horses and cattle after overwintering in 2004 and 2005. Within two weeks of the outbreak onset, 12,203 biting flies and 194 grasshoppers were collected near three equine-positive premises in Natrona County, WY. Insects were identified to the species level and tested by RT-qPCR for VSNJV polymerase (L) and phosphoprotein (P) gene RNA. Collected dipterans known to be competent for VSV transmission included Simulium black flies and Culicoides biting midges. VSNJV L and P RNA was detected in two pools of female Simulium bivittatum and subjected to partial genome sequencing. Phylogenetic analysis based on the hypervariable region of the P gene from black flies showed 100% identity to the isolate obtained from the index horse case on the same premises. This is the first report of VSNJV in S. bivittatum in WY and the first field evidence of possible VSV maintenance in black fly populations during an outbreak.

Detection Limits of Optical Gas Imaging for Natural Gas Leak Detection in Realistic Controlled Conditions.

Optical gas imaging (OGI) is a commonly utilized leak detection method in the upstream and midstream sectors of the U.S. natural gas industry. This study characterized the detection efficacy of OGI surveyors, using their own cameras and protocols, with controlled releases in an 8-acre outdoor facility that closely resembles upstream natural gas field operations. Professional surveyors from 16 oil and gas companies and 8 regulatory agencies participated, completing 488 tests over a 10 month period. Detection rates were significantly lower than prior studies focused on camera performance. The leak size required to achieve a 90% probability-of-detection in this study is an order-of-magnitude larger than prior studies. Study results indicate that OGI survey experience significantly impacts leak detection rate: Surveyors from operators/contractors who had surveyed more than 551 sites prior to testing detected 1.7 (1.5-1.8) times more leaks than surveyors who had completed fewer surveys. Highly experienced surveyors adjust their survey speed, examine components from multiple viewpoints, and make other adjustments that improve their leak detection rate, indicating that modifications of survey protocols and targeted training could improve leak detection rates overall.

Effects of Low-level Brodifacoum Exposure on the Feline Immune Response.

Anticoagulant rodenticides have been implicated as a potential inciting factor in the development of mange in wild felids, but a causative association between anticoagulant rodenticide exposure and immune suppression has not been established. Specific-pathogen-free domestic cats were exposed to brodifacoum over a 6-week period to determine whether chronic, low-level exposure altered the feline immune response. Cats were vaccinated with irrelevant antigens at different points during the course of the experiment to assess recall and direct immune responses. Measures of immune response included delayed-type hypersensitivity tests and cell proliferation assays. IgE and antigen-specific antibodies were quantified via ELISA assays, and cytokine induction following exposure to vaccine antigens was also analyzed. While cats had marked levels of brodifacoum present in blood during the study, no cats developed coagulopathies or hematologic abnormalities. Brodifacoum-exposed cats had transient, statistically significant decreases in the production of certain cytokines, but all other measures of immune function remained unaffected throughout the study period. This study indicates that cats may be more resistant to clinical effects of brodifacoum exposure than other species and suggests that the gross impacts of environmentally realistic brodifacoum exposure on humoral and cell-mediated immunity against foreign antigen exposures in domestic cats are minimal.

Evaluation of the Efficacy, Potential for Vector Transmission, and Duration of Immunity of MP-12, an Attenuated Rift Valley Fever Virus Vaccine Candidate, in Sheep.

Rift Valley fever virus (RVFV) causes serious disease in ruminants and humans in Africa. In North America, there are susceptible ruminant hosts and competent mosquito vectors, yet there are no fully licensed animal vaccines for this arthropod-borne virus, should it be introduced. Studies in sheep and cattle have found the attenuated strain of RVFV, MP-12, to be both safe and efficacious based on early testing, and a 2-year conditional license for use in U.S. livestock has been issued. The purpose of this study was to further determine the vaccine's potential to infect mosquitoes, the duration of humoral immunity to 24 months postvaccination, and the ability to prevent disease and viremia from a virulent challenge. Vaccination experiments conducted in sheep found no evidence of a potential for vector transmission to 4 North American mosquito species. Neutralizing antibodies were elicited, with titers of >1:40 still present at 24 months postvaccination. Vaccinates were protected from clinical signs and detectable viremia after challenge with virulent virus, while control sheep had fever and high-titered viremia extending for 5 days. Antibodies to three viral proteins (nucleocapsid N, the N-terminal half of glycoprotein GN, and the nonstructural protein from the short segment NSs) were also detected to 24 months using competitive enzyme-linked immunosorbent assays. This study demonstrates that the MP-12 vaccine given as a single dose in sheep generates protective immunity to a virulent challenge with antibody duration of at least 2 years, with no evidence of a risk for vector transmission.

Potential for North American mosquitoes (Diptera: Culicidae) to transmit rift valley fever virus.

To determine which arthropods should be targeted for control should Rift Valley fever virus (RVFV) be detected in North America, we evaluated Culex erraticus (Dyar and Knab), Culex erythrothorax Dyar, Culex nigripalpus Theobald, Culex pipiens L., Culex quinquefasciatus Say, Culex tarsalis Coquillett, Aedes dorsalis (Wiedemann), Aedes vexans (Meigen), Anopheles quadrimaculatus Say, and Culicoides sonorensis Wirth and Jones from the western, midwestern, and southern United States for their ability to transmit RVFV. Female mosquitoes were allowed to feed on adult hamsters inoculated with RVFV, after which engorged mosquitoes were incubated for 7-21 d at 260C, then allowed to refeed on susceptible hamsters, and tested to determine infection, dissemination, and transmission rates. Other specimens were inoculated intrathoracically, held for 7 d, and then allowed to feed on a susceptible hamster to check for a salivary gland barrier. When exposed to hamsters with viremias > or =10(8.8) plaque-forming units/ml blood, Cx. tarsalis transmitted RVFV efficiently (infection rate = 93%, dissemination rate = 56%, and estimated transmission rate = 52%). In contrast, when exposed to the same virus dose, none of the other species tested transmitted RVFV efficiently. Estimated transmission rates for Cx. erythrothorax, Cx. pipiens, Cx. erraticus, and Ae. dorsalis were 10, 8, 4, and 2%, respectively, and for the remaining species were < or = 1%. With the exception of Cx. tarsalis and Cx. pipiens, all species tested had moderate to major salivary gland barriers. None of the C. sonorensis became infected and none of the An. quadrimaculatus tested transmitted RVFV by bite, even after intrathoracic inoculation, indicating that these species would not be competent vectors of RVFV. Although Ae. vexans from Florida and Louisiana were relatively efficient vectors of RVFV, specimens of this species captured in Colorado or California were virtually incompetent, illustrating the need to evaluate local population for their ability to transmit a pathogen. In addition to laboratory vector competence, factors such as seasonal density, host feeding preference, longevity, and foraging behavior should be considered when determining the potential role that these species could play in RVFV transmission.

Blood-feeding behavior of vesicular stomatitis virus infected Culicoides sonorensis (Diptera: Ceratopogonidae).

To determine whether vesicular stomatitis virus (VSV) infection of Culicoides sonorensis Wirth & Jones (Diptera: Ceratopogonidae) affects subsequent blood-feeding behavior, midges injected with either virus-infected or virus-free cell lysates were allowed to blood feed for short (10-min) or long (60-min) periods on 2, 3, and 4 d postinoculation (DPI). Generalized linear mixed models were fit to test the effects of infection status, duration of feeding period, and DPI on the percentage of females that blood fed. VSV-infection significantly reduced the percentage of females that blood fed on 2 DPI, the day of peak virus titer. On 3 DPI a significantly greater percentage of midges blood fed when allowed 60 min to feed. This effect was not seen on 2 and 4 DPI and was not dependent on VSV infection status. The impact of changes in blood-feeding behavior by infected insects on virus transmission is discussed.

First records for Culex (Melanoconion) limacifer Komp and Culex (Melanoconion) dunni dyar from Guatemala.

Larvae of Culex (Melanoconion) limacifer Komp and Culex (Melanoconion) dunni Dyar were collected during June 2004 in Guatemala. All specimens were individually reared to the adult stage. Specimens were identified based upon examination of the male genitalia and characters of the associated larval and pupal exuviae. These are the first records of these 2 species in Guatemala.

A Rift Valley fever risk surveillance system for Africa using remotely sensed data: potential for use on other continents.

The authors developed a monitoring and risk mapping system using normalized difference vegetation index (NDVI) times series data derived from the advanced very high resolution radiometer (AVHRR) instrument on polar orbiting national oceanographic and atmospheric administration (NOAA) satellites to map areas with a potential for a Rift Valley fever (RVF) outbreaks in sub-Saharan Africa. This system is potentially an important tool for local, national and international organisations involved in the prevention and control of animal and human disease, permitting focused and timely implementation of disease control strategies several months before an outbreak. We are currently developing a geographic information system (GIS)-based remotely sensed early warning system for potential RVF vectors in the United States. Forecasts of the potential emergence of mosquito vectors will be disseminated throughout the United States, providing several months' warning in advance of potentially elevated mosquito populations. This would allow timely, targeted implementation of mosquito control, animal quarantine and vaccine strategies to reduce or prevent animal and human disease.

Association mapping of segregating sites in the early trypsin gene and susceptibility to dengue-2 virus in the mosquito Aedes aegypti.

Evidence suggests that midgut trypsins in Aedes aegypti condition the mosquito's ability to become infected with the dengue-2 flavivirus (DEN2). The activity of early trypsin protein peaks approximately 3 h after blood feeding and then drops within a few hours. We use association mapping to test the hypothesis that segregating sites in early trypsin condition midgut susceptibility to DEN2 virus. A total of 1642 females from throughout Mexico and the southern US were fed an artificial blood meal containing DEN2. After 2 weeks, mosquito heads and midguts were tested for DEN2. Mosquitoes with an infected head were classified as susceptible, those without a midgut infection had an infection barrier, and those with an infected gut but no head infection had an escape barrier. The early trypsin gene was amplified in two overlapping pieces from each mosquito and analyzed for single strand conformation polymorphisms (SSCPs). Unique SSCP genotypes were sequenced and 90 segregating sites were found. The dataset was divided into the four geographic regions within which Ae. aegypti is panmictic in Mexico. Heterogeneity chi2 analyses between alleles or genotypes and infection phenotypes demonstrated significant associations but allelic and genotypic effects were inconsistent among geographic regions. No consistent associations were found between segregating sites in early trypsin and susceptibility to DEN2 in Ae. aegypti in Mexico.

Effect of mosquito midgut trypsin activity on dengue-2 virus infection and dissemination in Aedes aegypti.

The effect of mosquito midgut trypsins in dengue serotype 2 flavivirus (DENV-2) infectivity to Aedes aegypti was studied. Addition of soybean trypsin inhibitor (STI) in a DENV-2 infectious blood meal resulted in a 91-97% decrease in midgut DENV-2 RNA copies (qRT-PCR analysis). STI treatment also resulted in slower DENV-2 replication in the midgut, less DENV-2 E protein expression, and decreased dissemination to the thorax and the head. A second uninfected blood meal, 7 days after the STI-treated infectious meal, significantly increased DENV-2 replication in the midgut and recovered oogenesis, suggesting that the lower viral infection caused by STI was in part due to a nutritional effect. Mosquitoes fed DENV-2 digested in vitro with bovine trypsin (before STI addition) exhibited a transient increase in midgut DENV-2 4 days postinfection. Blood digestion and possibly DENV-2 proteolytic processing, mediated by midgut trypsins, influence the rate of DENV-2 infection, replication, and dissemination in Ae. aegypti.

Selection of D2S3, an Aedes aegypti (Diptera: Culicidae) strain with high oral susceptibility to Dengue 2 virus and D2MEB, a strain with a midgut barrier to Dengue 2 escape.

Family-based phenotypic selection was used to breed two genetic strains of Aedes aegypti L. that differ in susceptibility to infection with dengue serotype 2 virus (DEN-2) strain JAM1409. A Dengue 2 Susceptible on 3 chromosomes (D2S3) strain was bred from Ae. aegypti aegypti and Ae. aegyptiformosus P1 parents to have a high midgut infection rate (MIR) and a high disseminated infection rate (DIR). A Dengue 2 Midgut Escape Barrier (D2MEB) strain was bred from D2S3 and Houston P1 parents to have a high MIR and a low DIR. After selection in the F2 generation, single strand conformation polymorphism genotypes were determined at cDNA marker loci throughout the genome to test for Mendelian ratios and thereby identify regions containing deleterious or lethal alleles. Both strains were orally challenged with two other DEN-2 genotypes, two DEN-1 genotypes, one DEN-3 genotype, and two DEN-4 genotypes. There were significant differences in MIR and DIR for the different virus strains in both the D2S3 and the D2MEB mosquito lines.

Quantitative trait loci that control dengue-2 virus dissemination in the mosquito Aedes aegypti.

The mosquito Aedes aegypti is the most important vector of yellow fever and dengue fever flaviviruses. Ae. aegypti eradication campaigns have not been sustainable and there are no effective vaccines for dengue viruses. Alternative control strategies may depend upon identification of mosquito genes that condition flavivirus susceptibility and may ultimately provide clues for interrupting transmission. Quantitative trait loci affecting the ability of Ae. aegypti to develop a dengue-2 infection in the midgut have been mapped previously. Herein we report on QTL that determine whether mosquitoes with a dengue-2-infected gut can then disseminate the virus to other tissues. A strain selected for high rates of dengue-2 dissemination was crossed to a strain selected for low dissemination rates. QTL were mapped in the F(2) and again in an F(5) advanced intercross line. QTL were detected at 31 cM on chromosome I, at 32 cM on chromosome II, and between 44 and 52 cM on chromosome III. Alleles at these QTL were additive or dominant in determining rates of dengue-2 dissemination and accounted for approximately 45% of the phenotypic variance. The locations of dengue-2 midgut infection and dissemination QTL correspond to those found in earlier studies.

Variation in vector competence for dengue 2 virus among 24 collections of Aedes aegypti from Mexico and the United States.

Aedes aegypti from 24 collections in Mexico and the United States were challenged orally with dengue 2 virus JAM1409 (DEN-2 JAM1409). The vector competence (VC) of the populations ranged from 24% to 83%. Mosquito populations from the Yucatan exhibited greater VC than those from other areas of Mexico. The presence or absence of a midgut infection barrier (MIB) and a midgut escape barrier (MEB) was determined for mosquitoes in each population. The percentage of mosquitoes exhibiting an MIB ranged from 14% to 59%, and those exhibiting an MEB ranged from 4% to 43% in the collections. The MIB and MEB were not completely independent as determined by regression analysis. Midgut infection rates were dose dependent.

Flavivirus susceptibility in Aedes aegypti.

Aedes aegypti is the primary vector of yellow fever (YF) and dengue fever (DF) flaviviruses worldwide. In this review we focus on past and present research on genetic components and environmental factors in Aedes aegypti that appear to control flavivirus transmission. We review genetic relationships among Ae. aegypti populations throughout the world and discuss how variation in vector competence is correlated with overall genetic differences among populations. We describe current research into how genetic and environmental factors jointly affect distribution of vector competence in natural populations. Based on this information, we propose a population genetic model for vector competence and discuss our recent progress in testing this model. We end with a discussion of approaches being taken to identify the genes that may control flavivirus susceptibility in Ae. aegypti.

Differential cytokine and antibody responses to adult and larval stages of Onchocerca volvulus consistent with the development of concomitant immunity.

The possibility of concomitant immunity and its potential mechanisms in Onchocerca volvulus infection were examined by analyzing cytokine and antibody responses to infective larval (third-stage larvae [L3] and molting L3 [mL3]), adult female worm (F-OvAg), and skin microfilaria (Smf) antigens in infected individuals in a region of hyperendemicity in Cameroon as a function of age. Peripheral blood mononuclear cell interleukin 5 (IL-5) responses to F-OvAg and Smf declined significantly with age (equivalent to years of exposure to O. volvulus). In contrast, IL-5 secretion in response to L3 and mL3 remained elevated with increasing age. Gamma interferon responses to L3, mL3, and F-OvAg were low or suppressed and unrelated to age, except for responses to Smf in older subjects. IL-10 levels were uniformly elevated, regardless of age, in response to L3, mL3, and F-OvAg but not to Smf, for which levels declined with age. A total of 49 to 60% of subjects had granulocyte-macrophage colony-stimulating factor responses to all O. volvulus antigens unrelated to age. Analysis of levels of stage-specific immunoglobulin G3 (IgG3) and IgE revealed a striking, age-dependent dissociation between antibody responses to larval antigens (L3 and a recombinant L3-specific protein, O. volvulus ALT-1) which were significantly increased or maintained with age and antibody responses to F-OvAg, which decreased. Levels of IgG1 to L3 and F-OvAg were elevated regardless of age, and levels of IgG4 increased significantly with age, although not to O. volvulus ALT-1, which may have unique L3-specific epitopes. Immunofluorescence staining of whole larvae showed that total anti-L3 immunoglobulin levels also increased with the age of the serum donor. The separate and distinct cytokine and antibody responses to adult and infective larval stages of O. volvulus which are age related are consistent with the acquisition of concomitant immunity in infected individuals.