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In November 2004, bluetongue virus (family Reoviridae, genus Orbivirus, BTV) serotype 1 (BTV-1) was detected for the first time in the United States from a hunter-killed deer in St. Mary Parish, LA. In 2005, sera surveys were conducted on three cattle farms near the area where the deer was found, and BTV-1-seropositive cattle were found on two of the three farms; in 2006, sera surveys from the cattle on the three farms did not detect any BTV-1-positive animals. The purpose of this study was to survey ceratopogonid populations at the three farms and test field-collected specimens for the presence of BTV and epizootic hemorrhagic disease virus (family Reoviridae, genus Orbivirus, EHDV). Miniature CDC light traps and New Jersey traps were used to capture ceratopogonids on the three farms from January 2006 through November 2007. In total, 3,319 ceratopogonids were captured, including 1,790 specimens of 10 different species of Culicoides. IR-RT-polymerase chain reaction (PCR) was performed to screen for BTV and EHDV in 264 pools representing 2,309 specimens collected at the farms. All positive samples were sequenced for serotype determination. Five pools of 275 (1.8%) were positive for BTV. Pools of four species of Culicoides were found to be positive: Culicoides crepuscularis (Malloch), Culicoides debilipalpis Lutz (two pools), Culicoides haematopotus Malloch, and Gulicoidesfurens (Poey). The amplicons of the positive specimens were sequenced and found to be identical to both BTV-17 and BTV-13. During our study, no BTV-1 transmission was detected in cattle, and no BTV-1 was detected in specimens of ceratopogonids.

A TissueLyser system (QIAGEN) was used to rapidly and accurately estimate bluetongue virus "loads" in individual adult Culicoides sonorensis Wirth & Jones (Diptera: Ceratopogonidae). The optimized homogenization program that was developed, involved shaking insects for 1 min at 25 Hz with 2- or 3-mm stainless steel ball bearings. This program was used to measure the quantities of bluetongue virus present in insects that had either been inoculated or had ingested a viremic bloodmeal through an artificial membrane. The virus titers obtained using either infection technique and the optimized program did not differ significantly from those obtained using a polypropylene motor-driven pestle, a method that is currently in common use for studies of vector competence). The advantages of the new method, as a rapid means of detecting fully disseminated infections in individual field-caught flies, are discussed. Its use is compared with the processing of pools of Culicoides by conventional methods, where the extent of dissemination of the virus is unknown and could wrongly implicate species that are of low importance in transmission.

Oral susceptibility to infection with bluetongue virus (family Resviridae, genus Orbivirus, BTV) serotype 9 was characterized in three Palaearctic species of Culicoides (Diptera: Ceratopogonidae). Variation in susceptibility to infection by using a recently described feeding technique was shown to occur between populations of Culicoides obsoletus Meigen complex midges from different geographic regions of the United Kingdom with virus infection rates varying from 0.4 to 7.4% of those tested. Susceptibility to infection was consistent on an annual basis at selected sites. Prevalence of infection in the most susceptible populations of both the C. obsoletus and Culicoides pulicaris L. complexes was comparable with that of Culicoides imicola Kieffer, the major vector of BTV in southern Europe and throughout Africa, when using the same feeding method and virus. These results are discussed with reference to the potential threat of the virus to susceptible livestock in northern Europe.

Biting midges of the genus Culicoides are important in the transmission of viral diseases affecting wild and domestic ungulates, including bluetongue (BLU) and epizootic hemorrhagic disease (EHD). The primary known vector for these viruses is C. sonorensis Wirth & Jones, however, it has been speculated that other species of Culicoides may also be involved. One potential candidate is C. mohave, a poorly studied species found in inland desert areas of the southwestern United States. In 2000 and 2001, we collected C. mohave and C. sonorensis at six sites in a previously unsurveyed area in the Sonoran Desert of southwestern Arizona and used PCR to detect nucleic acids associated with BLU and EHD viruses. C. mohave was abundant at two low-elevation sites on the study area, but uncommon or absent elsewhere. C. sonorensis commonly occurred along with C. mohave at one site, but was much less abundant. All C. mohave pools were negative for BLU viral RNA, however, 35% yielded positive results for EHD. All C. sonorensis were negative for both BLU and EHD. Our results suggest that C. mohave is a potential vector of EHD virus in this area, however additional studies are needed to determine its ability to transmit EHD.

Vectorial capacity of Culicoides sonorensis Wirth & Jones for the transmission of bluetongue (BLU) virus was examined at a southern California dairy from January 1995 to December 1997. Insects were collected one to two times per week in five CDC-type suction traps (without light) baited with CO2 at a constant release rate of 1,000 ml/min. BLU virus was detected in midges collected from May through December with an estimated overall infection rate of 0.08%. The BLU virus infection rate of field-captured midges was not correlated with sentinel calf seroconversions to BLU virus. Sentinel calf seroconversions were highly seasonal, occurring from August through November with most calves seroconverting during September and October. Vector competence of field-collected nulliparous flies fed a locally acquired serotype of BLU virus in the laboratory was stable among years (17-23%). Vectorial capacity was strongly correlated with BLU virus transmission (measured by sentinel calf seroconversions) during 1995 and 1996, but not during 1997. Host biting rate estimated for traps nearest to the sentinel calves was the index best correlated with BLU virus transmission for all study years and was most highly correlated with sentinel seroconversions 4 wk later. The utility of vectorial capacity and its component variables is discussed for this system.

Immature Culicoides variipennis (Coquillett) were sampled from aquatic habitats throughout Virginia, reared to adults, and examined by isozyme electrophoresis to assess their taxonomic status. Data from 22 counties showed that C. v. variipennis is widespread and common, the predominant taxon throughout Virginia, and genetically similar to C. v. variipennis in Maryland. Because C. v. variipennis is considered an inefficient vector of the bluetongue viruses, this observation is consistent with the low seroprevalence of bluetongue in indigenous livestock of the mid-Atlantic region. Culicoides v. sonorensis Wirth & Jones, considered to be the primary North American vector of the bluetongue viruses, was recovered in large numbers only from a wastewater lagoon at a dairy in southeastern Virginia, but also was detected at low levels in 6 other counties. Comparison of genetic distances and patterns of discriminating alleles among Virginia populations of C. v. variipennis and C. v. sonorensis showed that respective subspecies are genetically distinct and show no evidence of introgression, irrespective of geographic- and habitat-level sympatry. The persistence of a pure C. v. sonorensis population in a dairy wastewater lagoon may reflect physico-chemical factors that influence the distribution of immature C. variipennis complex populations. A better understanding of the distribution of the C. variipennis complex will benefit regionalization of U.S. exports of livestock and livestock germplasm to bluetongue-free countries.

An avidin-biotin complex (ABC) dot-blot, an antigen capture enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy (IEM) were used to detect bluetongue (BLU) virus and viral antigen in field-collected C. varriipennis sonorensis Wirth & Jones from an enzootic BLU area in northeastern Colorado. This is the 1st attempt to apply these immunodiagnostic methods to an epidemiologically relevant, large-scale ecological system. One of the 1,800 midges (0.0005%) was positive by the dot-blot procedure, 2 (0.0011%) were positive by the ELISA, and BLU virus was identified in 8 midges (0.0044%) by IEM. These data are interpreted in context of the "whole system" of the disease to provide a framework for determining the knowledge gaps in our understanding and directing future studies in these areas. Our basic model of BLU ecology suggests that the infection rates found by the diagnostic methods are within expected ranges, thus strongly supporting the proposed ecological model and the work used to parameterize the model. This integration of immunodiagnostic methods and ecology makes it evident that further investigations of daily mortality during the extrinsic incubation period are vital to a better understanding of BLU virus occurrence in Culicoides vector and vertebrate host populations.

An avidin-biotin complex (ABC) dot-blot, an antigen capture enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy (IEM) were used to detect bluetongue (BLU) virus or viral antigen or both in adult Culicoides variipennis sonorensis Wirth & Jones. The dot-blot and ELISA procedures detected viral antigen in 10-22% (depending on serotype) of the biting midges infected with BLU-2, BLU-10, BLU-13, and BLU-17 and approximately 68% of the midges infected with BLU-11. IEM analyses revealed BLU virus in salivary glands, fat body, and thoracic muscle tissue from infected insects. There appeared to be selective growth of the virus in salivary gland tissue.

To help determine specific vectors of epizootic hemorrhagic disease (EHD) and bluetongue (BT) viruses for white-tailed deer, Odocoileus virginianus Zimmermann, in the southeastern United States, Culicoides sp. midges were collected during epizootics of hemorrhagic disease among captive white-tailed deer in Georgia, Mississippi, and North Carolina. Culicoides variipennis (Coquillett), a confirmed vector of EHD and BT viruses, was present in low numbers in light-trap collections made at all sites. Collections from deer made in Georgia and North Carolina yielded only a single specimen of C. variipennis. Other Culicoides species present in far greater numbers during the epizootics included C. lahillei Lutz, C. paraensis (Goeldi), and C. stellifer (Coquillett) C. lahillei warrants particular attention as a potential vector because its readily feeds on white-tailed deer and was by far the predominant species collected from deer during the epizootics.

Field collected Culicoides brevitarsis Kieffer and C. wadai Kitaoka were fed on sheep that had been artificially infected with a field-isolate of either bluetongue virus serotype 3 (BLU3), BLU9, BLU16, or BLU23. Feeding rates averaged 11.9% but were variable. Survival of midges during incubation tended to be enhanced by the addition of antibiotics and fungicide to the diet. Attempts to transmit virus to sheep by the bite of these two species were unsuccessful. Both C. brevitarsis and C. wadai had infection rates of between 1.3 and 1.5% for BLU16 and BLU23. The infection rate for BLU3 in C. wadai was low at 0.07% whereas those for BLU3 in C. brevitarsis and BLU9 in both species could not be calculated because of insufficient virus isolations. The relatively high infection rates for BLU16 and BLU23 suggest that both C. brevitarsis and C. wadai are capable of circulating these viruses in the field.

The transmission of bluetongue virus (BLU) to sheep and cattle relative to Culicoides abundance and infection was studied in two areas of the Louisiana State University Agricultural Center, Baton Rouge, LA. The seroprevalence of BLU in beef cattle was 70.5% in 1989 and 37.0% in 1990; 2 of 37 animals tested in both years became positive, indicating active transmission. The prevalence of BLU in sheep decreased from 52.2% in 1989 to 34% in 1990; seroconversion was not detected. Serotypes of positive sheep were as follows: 13 sheep were positive for BLU-13, 6 for BLU-17, 2 for BLU-2, and 2 for both BLU-13 and BLU-17. The seroprevalence of BLU virus in dairy bulls increased from 15.4% in 1989 to 47.4% in 1990; 0 and 7 seroconversions occurred in 1989 and 1990, respectively. Serotypes of BLU from the bulls were as follows: five bulls for BLU-13; two for BLU-17; one for BLU-13 and BLU-17; and one for BLU-13, BLU-17, and BLU-2. Peaks in the abundance of Culicoides variipennis (Coquillett) and C. stellifer (Coquillett) collected in New Jersey light traps corresponded with the suspected transmission dates during 1990. Bluetongue viral RNA was detected in one of 381 pools (6,072 flies) of C. variipennis; no virus was detected in 125 pools (884 flies) of C. stellifer.

Culicoides boydi new species is described from Riverside County, CA. A taxonomic key, table of numerical characters, and female wing photographs are presented to distinguish it from the three other species of the Culicoides pusillus species group of the subgenus Avaritia. Brief notes are presented on its biology and distribution and suspected vector potential in the transmission of bluetongue virus to desert bighorn sheep in California.

An enzyme-linked immunoassay for detecting bluetongue virus in infected Culicoides variipennis was evaluated using a nested analysis of variance to determine sources of experimental error in the procedure. The major source of variation was differences among individual insects (84% of the total variance). Storing insects at -70 degrees C for two months contributed to experimental variation in the ELISA reading (14% of the total variance) and should be avoided. Replicate assays of individual insects were shown to be unnecessary, since variation among replicate wells and plates was minor (2% of the total variance).

An antigen capture enzyme-linked immunosorbent assay (ELISA) was developed to detect bluetongue virus (BTV) in the vector Culicoides variipennis (Coquillett). This ELISA used polyclonal and monoclonal antibodies in a biotin-avidin immunoperoxidase system to detect BTV in individual adult female flies that had taken an infectious blood meal. Detection of BTV with the ELISA correlated with detection of BTV from the same flies by plaque assay in cell culture. The ELISA was group-specific for the five U.S. serotypes of BTV and did not cross react with closely related epizootic hemorrhagic disease virus.

Monitoring infection rates of Culicoides variipennis (Coquillett) with bluetongue virus in rangeland deer and cattle often requires prolonged field excursions. Methodology that fits field processing and storage was evaluated under controlled laboratory conditions. Triethylamine was used to anesthetize Culicoides for sorting. A portable liquid nitrogen vapor shipping container offered a convenient means of insect storage at temperatures that ensured preservation of the virus and eliminated fungal and bacterial contamination of cell cultures. Triethylamine and liquid nitrogen storage had little effect on recovery of bluetongue virus from experimentally infected C. variipennis and sheep blood stored under similar conditions.

The avidin-biotin complex immunoperoxidase system has been used with nitrocellulose membranes to detect bluetongue virus serotypes 10 (BTV-10) and 11 (BTV-11) and epizootic hemorrhagic disease virus (EHDV) serotype 1 (EHDV-1) in individual specimens of Culicoides variipennis (Coquillett). The cross reactivity between BTV-10 and BTV-11 indicates that polyclonal antisera cannot be used to distinguish between flies infected with BTV-10 and BTV-11; however, the procedure can be used to discriminate flies infected with BTV-10 or BTV-11 from flies infected with EHDV-1. Because the test is rapid and sensitive and does not require expensive equipment, it could be used in the field to determine the relative numbers of flies infected with BTV or EHDV.