Diagnostic applications of molecular and serological assays for bluetongue and African horse sickness.

The availability of rapid, highly sensitive and specific molecular and serologic diagnostic assays, such as competitive enzyme-linked immunosorbent assay (cELISA), has expedited the diagnosis of emerging transboundary animal diseases, including bluetongue (BT) and African horse sickness (AHS), and facilitated more thorough characterisation of their epidemiology. The development of assays based on real-time, reverse-transcription polymerase chain reaction (RT-PCR) to detect and identify the numerous serotypes of BT virus (BTV) and AHS virus (AHSV) has aided in-depth studies of the epidemiology of BTV infection in California and AHSV infection in South Africa. The subsequent evaluation of pan-serotype, real-time, RT-PCR-positive samples through the use of serotype-specific RT-PCR assays allows the rapid identification of virus serotypes, reducing the need for expensive and time-consuming conventional methods, such as virus isolation and serotype-specific virus neutralisation assays. These molecular assays and cELISA platforms provide tools that have enhanced epidemiologic surveillance strategies and improved our understanding of potentially altered Culicoides midge behaviour when infected with BTV. They have also supported the detection of subclinical AHSV infection of vaccinated horses in South Africa. Moreover, in conjunction with whole genome sequence analysis, these tests have clarified that the mechanism behind recent outbreaks of AHS in the AHS-controlled area of South Africa was the result of the reversion to virulence and/or genome reassortment of live attenuated vaccine viruses. This review focuses on the use of contemporary molecular diagnostic assays in the context of recent epidemiologic studies and explores their advantages over historic virus isolation and serologic techniques.

La disponibilité d'essais diagnostiques moléculaires et sérologiques rapides, hautement sensibles et spécifiques tels que l'épreuve immuno-enzymatique de compétition (ELISAc), a accéléré le diagnostic des maladies animales transfrontalières émergentes, dont la fièvre catarrhale ovine (FCO) et la peste équine, et contribué à dresser un tableau épidémiologique plus complet de ces maladies. Grâce à la mise au point d'essais basés sur l'amplification en chaîne par polymérase en temps réel couplée à une transcription inverse (RT­PCR) qui permettent de détecter et d'identifier les nombreux sérotypes du virus de la fièvre catarrhale du mouton et du virus de la peste équine, des études approfondies ont pu être conduites sur l'épidémiologie de l'infection par le virus de la fièvre catarrhale du mouton en Californie et de l'infection par le virus de la peste équine en Afrique du Sud. L'évaluation postérieure des échantillons positifs à une RT­PCR en temps réel de groupe (détectant le virus quel que soit le sérotype) au moyen de RT­PCR spécifiques de chaque sérotype permet d'identifier rapidement le sérotype causal et de limiter le recours à des méthodes classiques onéreuses et chronophages comme l'isolement viral ou les essais de neutralisation virale spécifiques de chaque sérotype. Les outils fournis par ces essais moléculaires et par les plateformes ELISAc ont renforcé les stratégies de surveillance épidémiologique et permis de mieux connaître les altérations potentielles de comportement chez les tiques Culicoides infectées par le virus de la fièvre catarrhale du mouton. Ils ont également contribué à détecter les cas d'infection asymptomatique par le virus de la peste équine chez des chevaux vaccinés en Afrique du Sud. En outre, associés avec l'analyse de séquences du génome entier, ces tests ont révélé que le mécanisme sous-jacent aux récents foyers de peste équine dans la zone de contrôle en Afrique du Sud correspondait à une réversion vers la virulence et/ou à un réassortiment du génome des souches de vaccin à virus vivant atténué. Les auteurs passent en revue l'utilisation des essais de diagnostic moléculaire de nouvelle génération dans le contexte de récentes études épidémiologiques et cherchent à établir leurs avantages par rapport aux techniques classiques d'isolement viral et de recherche sérologique.

La existencia de ensayos moleculares y serológicos de diagnóstico rápidos y de gran sensibilidad y especificidad, como el ensayo inmunoenzimático de competición (ELISAc), ha acelerado el diagnóstico de enfermedades animales transfronterizas emergentes, como la lengua azul o la peste equina, y facilitado una caracterización más exhaustiva de su epidemiología. La creación de ensayos basados en la reacción en cadena de la polimerasa acoplada a transcripción inversa (RT?PCR) en tiempo real para detectar y caracterizar los numerosos serotipos de los virus de la lengua azul y la peste equina ha ayudado a estudiar a fondo la epidemiología de sendos episodios infecciosos causados por el virus de la lengua azul en California y por el virus de la peste equina en Sudáfrica. El subsiguiente análisis de las muestras positivas a la prueba de RT?PC en tiempo real de cualquier serotipo con empleo de ensayos RT?PCR dirigidos específicamente contra uno u otro serotipo permite identificar rápidamente los serotipos víricos, lo que hace menos necesario el uso de métodos convencionales más caros y largos, como el aislamiento del virus o técnicas de neutralización vírica adaptadas específicamente a un serotipo. Estos dispositivos de ensayo molecular o de ELISAc ponen a nuestra disposición herramientas que potencian las estrategias de vigilancia epidemiológica y ayudan a conocer mejor las eventuales alteraciones del comportamiento de los jejenes Culicoides al ser infectados por el virus de la lengua azul. Estas técnicas han ayudado también a detectar en Sudáfrica casos de infección asintomática por el virus de la peste equina en caballos vacunados. Estas pruebas, además, empleadas en combinación con el análisis de secuencias genómicas completas, han servido para aclarar que el mecanismo subyacente a los recientes brotes de peste equina surgidos en la zona de Sudáfrica donde la enfermedad estaba bajo control fue fruto de la reversión a la virulencia y/o el reordenamiento genómico de virus vacunales atenuados. Los autores, centrándose en el uso de modernos ensayos moleculares de diagnóstico como parte de recientes estudios epidemiológicos, examinan las ventajas que ofrecen en comparación con las tradicionales técnicas serológicas y de aislamiento vírico.

Dividing the pie: differential dung pat size utilization by sympatric Haematobia irritans and Musca autumnalis.

Horn flies [Haematobia irritans (Diptera: Muscidae) (L.)] and face flies [Musca autumnalis (Diptera: Muscidae) De Geer] use the same larval resource, but their interactions are poorly studied. Dung pats (n = 350) were core sampled in the summers of 2012 and 2013 from irrigated pastures in Pomona, California, U.S.A. (34°03'N, 117°48'W) and held for face fly and horn fly emergence. Surface areas and estimated weights were recorded for each whole pat. Almost half (42.0%) of the pat cores yielded neither fly, 29.7% yielded horn flies only, 12.9% yielded face flies only and 15.4% yielded both flies. Of the fly-positive pats, surface area and mass were larger for face fly-occupied pats, whereas horn fly-occupied pats were smaller. Pats shared by the two species were intermediate. Horn flies per positive core were unaffected by the absence/presence of face flies, but half as many face flies emerged when pats were co-inhabited by horn flies. Face flies inhabited larger pats, which might better resist heating and drying, to which they are susceptible; horn flies inhabited a broad pat size range. Horn fly tolerance of lower dung moisture probably allows horn flies to colonize and survive in a wide range of pats in dry areas like southern California.

Trap placement and attractant choice affect capture and create sex and parity biases in collections of the biting midge, Culicoides sonorensis.

Culicoides sonorensis Wirth & Jones (Diptera: Ceratopogonidae) is the primary North American vector of bluetongue virus (BTV), which can cause high morbidity and mortality in ruminant livestock or wildlife. Worldwide, most Culicoides surveillance relies on light (usually UV) traps typically placed near animals or larval development sites. However, the trapping method can cause sex, species and parity biases in collections. We collected C. sonorensis from three dairies in California using suction traps baited with CO2 , UV light or CO2 + UV placed near animals, wastewater ponds, or in fields. Higher numbers of parous females were collected using CO2 + UV traps, although this difference was only significant on one dairy. UV traps were poor at collecting nulliparous females, but the addition of UV to a trap increased the abundance of males in a collection. Traps set in open fields collected significantly higher numbers of males and females than in either of the other two locations. In some cases, there was a significant interaction between the trap type and site. We discuss the limitations of traditional trapping methodologies for C. sonorensis and make suggestions for vector surveillance.

Bionomics of temperate and tropical Culicoides midges: knowledge gaps and consequences for transmission of Culicoides-borne viruses.

Culicoides midges are abundant hematophagous flies that vector arboviruses of veterinary and medical importance. Dramatic changes in the epidemiology of Culicoides-borne arboviruses have occurred since 1998, including the emergence of exotic viruses in northern temperate regions, increases in global disease incidence, and enhanced virus diversity in tropical zones. Drivers may include changes in climate, land use, trade, and animal husbandry. New Culicoides species and new wild reservoir hosts have been implicated in transmission, highlighting the dynamic nature of pathogen-vector-host interactions. Focusing on potential vector species worldwide and key elements of vectorial capacity, we review the sensitivity of Culicoides life cycles to abiotic and biotic factors. We consider implications for designing control measures and understanding impacts of environmental change in different ecological contexts. Critical geographical, biological, and taxonomic knowledge gaps are prioritized. Recent developments in genomics and mathematical modeling may enhance ecological understanding of these complex arbovirus systems.

Lack of Evidence for Laboratory and Natural Vertical Transmission of Bluetongue Virus in Culicoides sonorensis (Diptera: Ceratopogonidae).

Culicoides sonorensis (Wirth & Jones) is the principal North American vector of bluetongue virus (BTV). BTV infection of livestock is distinctly seasonal (late summer and fall) in temperate regions of the world such as California, which has led to speculation regarding vertical transmission of the virus within the midge vector as a potential mechanism for interseasonal maintenance ("overwintering") of the virus. To evaluate potential vertical transmission of BTV in its midge vector, we fed adult midges BTV-spiked blood and used a BTV-specific quantitative reverse transcriptase polymerase chain reaction assay to evaluate parent, egg, and progeny stages of laboratory-reared C. sonorensis for the presence of viral nucleic acid. Whereas BTV nucleic acid was weakly detected in egg batches of virus-fed female midges, virus was never detected in subsequent progeny stages (larvae, pupae, and F1 generation adults). Similarly, BTV was not detected in pools of larvae collected from the waste-water lagoon of a BTV-endemic dairy farm in northern California during the seasonal period of virus transmission. Collectively, these results indicate that BTV is not readily transmitted vertically in C. sonorensis, and that persistence of the virus in long-lived parous female midges is a more likely mechanism for overwintering of BTV in temperate regions.

Estimating Culicoides sonorensis biting midge abundance using digital image analysis.

ImageJ is an open-source software tool used for a variety of scientific objectives including cell counting, shape analysis and image correction. This technology has previously been used to estimate mosquito abundance in surveillance efforts. However, the utility of this application for estimating abundance or parity in the surveillance of Culicoides spp. (Diptera: Ceratopogonidae) has not yet been tested. Culicoides sonorensis (Wirth and Jones), a biting midge often measuring 2.0-2.5 mm in length, is an economically important vector of ruminant arboviruses in California. Current surveillance methods use visual sorting for the characteristics of midges and are very time-intensive for large studies. This project tested the utility of ImageJ as a tool to assist in gross trap enumeration as well as in parity analysis of C. sonorensis in comparison with traditional visual methods of enumeration using a dissecting microscope. Results confirmed that automated counting of midges is a reliable means of approximating midge numbers under certain conditions. Further evaluation confirmed accurate and time-efficient parity analysis in comparison with hand sorting. The ImageJ software shows promise as a tool that can assist and expedite C. sonorensis surveillance. Further, these methods may be useful in other insect surveillance activities.

Assessment of survival and body size variation of Culicoides imicola (Diptera: Ceratopogonidae) as functions of "Candidatus Cardinium" (Bacteroidetes) infection status.

"Candidatus Cardinium hertigii" (Bacteroidetes) is a maternally inherited endosymbiont known from several arthropods. Its mechanisms for persistence in host populations are mostly reproductive manipulation, though it has been occasionally reported to improve fitness parameters in several hosts. In Culicoides (Diptera: Ceratopogonidae) biting midges, the prevalence of "Candidatus Cardinium" infection was documented as moderate, with no detectable sex bias. We therefore investigated whether "Candidatus Cardinium" affects important fitness parameters, such as survival and body size, in Culicoides imicola, a dominant vector species. Field-collected midges were trapped and analyzed for survival under different environmental conditions and antibiotic treatment, taking into account "Candidatus Cardinium" infection status and parity status (i.e., parous or nulliparous). Additionally, wing lengths were measured as a proxy parameter for body size and analyzed together with "Candidatus Cardinium" infection data. The findings revealed no difference in survival of Culicoides infected with "Candidatus Cardinium" and that of uninfected midges in both parity states and under all tested conditions: optimal, starvation, heat, and antibiotic treatment. Beyond survival, no wing length difference was found for "Candidatus Cardinium"-infected versus uninfected midges. In aggregate, these findings support our conclusion that "Candidatus Cardinium" does not have an overt effect on the survival and size of adult C. imicola midges. "Candidatus Cardinium" may affect immature stages or may alter adult reproductive performance.

Gonotrophic development and survival in field populations of Musca domestica (Diptera: Muscidae) at dairies in California, Minnesota, and Georgia, and the relationship of fly age to relative abundance of (Z)-9-tricosene (muscalure).

Adult female Musca domestica L. were collected in 2004 and 2005 from dairies in California, Minnesota, and Georgia. Relative abundance of (Z)-9-tricosene (muscalure) among the dominant eight hydrocarbons was determined. Fly heads then were removed to quantify pterin levels and estimate fly age, abdomens were dissected to score gonotrophic development and parity (follicular relics), and spermathecae were examined for sperm. Daily survival was assessed using two estimates of time required to become gravid: laboratory-based degree-day (DD) estimates and estimates based on pterin values in field-collected flies matched to their stages of gonotrophic development. Among newly emerged females (oocyte stage 1) with detectable muscalure, it comprised < approximately 1.5% of cuticular hydrocarbons. In muscalure-positive flies, muscalure comprised a higher proportion of cuticular hydrocarbons in older flies from California and Minnesota (6-9% when gravid) versus flies from Georgia (<2% when gravid). Females mated in early-intermediate stages of egg development. Life expectancy, using laboratory-derived estimates of time needed to become gravid, ranged from 3.6 to 10.6 d. Using equivalent pterin-based time estimates, life expectancy ranged from 4.0 to 19.5 d. Mean DD ages (12 degrees C threshold) of gravid flies varied widely (53-95 DD) and were congruent with laboratory-based estimates (52-57 DD) in only 7 of 12 farm-year combinations. Thus, house flies under natural conditions often required more time to develop eggs than laboratory models would predict, extending daily survival estimates based on gonotrophic age by 11-74%.

Housing and dustbathing effects on northern fowl mites (Ornithonyssus sylviarum) and chicken body lice (Menacanthus stramineus) on hens.

Hen housing (cage or cage-free) did not impact overall abundances of northern fowl mites, Ornithonyssus sylviarum (Canestrini & Fanzago) (Acari: Macronyssidae), or chicken body lice, Menacanthus stramineus (Nitzsch) (Phthiraptera: Menoponidae). Cage-free hens received a dustbox with sand plus diatomaceous earth (DE), kaolin clay or sulphur. Weekly use varied from none to 100% of hens; 73% of hens used the dustbox at least once. Ectoparasite populations on dustbathing hens (users) were compared with those on non-user cage-free and caged hens. All materials reduced ectoparasites on user hens by 80-100% after 1 week of dustbox use. Diatomaceous earth and kaolin failed to reduce ectoparasites on non-user hens, and ectoparasites on user hens recovered after dustbox removal. A sulphur dustbox eliminated mites from all hens (including non-users) within 2-4 weeks. Residual sulphur controlled mites until the end of the experiment (up to 9 weeks), even after the dustbox was removed. Louse populations on hens using the sulphur dustbox were reduced in 1-2 weeks. Residual sulphur effects were less evident in lice, but the use of a sulphur dustbox by a higher proportion of hens extended louse control to all hens. This is the first experimental study to show that bird dustbathing in naturally and widely available dust materials (particularly kaolin) can suppress ectoparasites and thus the behaviour is probably adaptive.

Responses of Ornithonyssus sylviarum (Acari: Macronyssidae) and Menacanthus stramineus (Phthiraptera: Menoponidae) to gradients of temperature, light, and humidity, with comments on microhabitat selection on chickens.

Responses of the northern fowl mite (NFM), Ornithonyssus sylviarum (Canestrini & Fanzago) (Acari: Macronyssidae), and the chicken body louse (CBL), Menacanthus stramineus (Nitzsch) (Phthiraptera: Menoponidae), to variation in temperature, light, and humidity were assessed in bioassays. The location on a continuous thermal gradient at which each ectoparasite arrested was recorded and analyzed. NFM adults arrested at an average temperature of 30.09 +/- 0.34 degrees C. Adult CBL and first-instar CBL nymphs arrested at 33.69 +/- 0.20 degrees C and 34.99 +/- 0.26 degrees C, respectively. Groups of each ectoparasite were placed into clear glass vials (n = 10/vial) with one half shaded, and vials were exposed to three light levels, as follows: high (200 micromolm(-2)s(-1)), low (4 micromolm(-2)s(-1)), and nearly no light (0 micromolm(-2)s(-1)). The vial cap edges provided an opportunity to assess the interactive effect of light with harborage. NFM avoided light and sought harborage. In low light, the harborage preference overrode the tendency to avoid light. CBL avoided the harborage and showed a minimal preference for light. A four-level humidity gradient was established in two separate experimental arenas for NFM and CBL. Trials were run in ambient light (4 micromolm(-2)s(-1)) for the NFM and in nearly no light for the CBL. The NFM gradient used 38 +/- 2%, 54 +/- 7%, 73 +/- 3%, and 90 +/- 4% RH, whereas the CBL gradient used 42 +/- 5%, 48 +/- 7%, 63 +/- 4%, and 73 +/- 5% RH. NFM showed no humidity response in the walking bioassay, but the CBL settled at the lowest humidity level. Temperature and humidity on different hen body regions were related to the bioassay results and observed on-host ectoparasite distributions.

Hen welfare in different housing systems.

Egg production systems have become subject to heightened levels of scrutiny. Multiple factors such as disease, skeletal and foot health, pest and parasite load, behavior, stress, affective states, nutrition, and genetics influence the level of welfare hens experience. Although the need to evaluate the influence of these factors on welfare is recognized, research is still in the early stages. We compared conventional cages, furnished cages, noncage systems, and outdoor systems. Specific attributes of each system are shown to affect welfare, and systems that have similar attributes are affected similarly. For instance, environments in which hens are exposed to litter and soil, such as noncage and outdoor systems, provide a greater opportunity for disease and parasites. The more complex the environment, the more difficult it is to clean, and the larger the group size, the more easily disease and parasites are able to spread. Environments such as conventional cages, which limit movement, can lead to osteoporosis, but environments that have increased complexity, such as noncage systems, expose hens to an increased incidence of bone fractures. More space allows for hens to perform a greater repertoire of behaviors, although some deleterious behaviors such as cannibalism and piling, which results in smothering, can occur in large groups. Less is understood about the stress that each system imposes on the hen, but it appears that each system has its unique challenges. Selective breeding for desired traits such as improved bone strength and decreased feather pecking and cannibalism may help to improve welfare. It appears that no single housing system is ideal from a hen welfare perspective. Although environmental complexity increases behavioral opportunities, it also introduces difficulties in terms of disease and pest control. In addition, environmental complexity can create opportunities for the hens to express behaviors that may be detrimental to their welfare. As a result, any attempt to evaluate the sustainability of a switch to an alternative housing system requires careful consideration of the merits and shortcomings of each housing system.

Beak condition and cage density determine abundance and spatial distribution of northern fowl mites, Ornithonyssus sylviarum, and chicken body lice, Menacanthus stramineus, on caged laying hens.

Adult White Leghorn hens (Hy-Line strain W-36) were inoculated with either northern fowl mites or chicken body lice, and the ectoparasite populations were monitored over periods of 9 to 16 wk. Two beak conditions (beak trimmed or beak intact) and 2 housing densities (1 or 2 hens per 25 × 31 cm suspended wire cage) were tested. Populations of both ectoparasites were at least 10 times lower on beak-intact hens compared with populations on beak-trimmed hens. Cage density did not influence mite numbers, but higher numbers of lice (2 to 3 times) developed on hens held at the higher cage density. Louse distribution on the body and louse population age structure were also influenced by host beak condition. Beak-intact hens had a higher proportion of lice under the wings, whereas beak-trimmed hens had the majority of lice on the lower abdomen. Louse populations on beak-trimmed hens also comprised relatively more immature stages than populations found on beak-intact hens. The effects are likely related to decreased grooming efficiency by beak-trimmed hens and, in the case of lice, the higher host density. The high mite and louse populations on most commercial caged laying hens are probably a direct result of beak trimming. However, selection of more docile breeds that can be held without trimming may allow the hens themselves to reduce ectoparasites below economically damaging levels. This could benefit producers, animal welfare advocates, and human health by reducing 1) costs of beak trimming, 2) pesticide treatment costs (including human and bird chemical exposure concerns), and 3) objections to beak trimming from the animal welfare community.

Characterization of age and cuticular hydrocarbon variation in mating pairs of house fly, Musca domestica, collected in the field.

House flies, Musca domestica L., were collected in copula over two summers from six dairies located in three climatically distinct regions in the U.S.A. southern California, Minnesota and Georgia. Ages of males and females from a total of 511 mating pairs were estimated using pterin analysis. Cuticular hydrocarbon profiles and gonotrophic ages of females also were evaluated. Mean age of mating males ranged from 54 to 102 degree-days (DD) (4-10 days based on field air temperatures), depending on the farm. Very young males (< 10-20 DD) and old males (> 200 DD) were rare in mating pairs. Mean female age at mating ranged from 20 to 46 DD (2.5-4 days). All mating females had eggs in the early stages of vitellogenesis and 99.2% were nulliparous. However, some older and parous females were collected, demonstrating that re-mating can occur in the field. Head width measurements of mating pairs suggested that assortative mating by size did not occur. The cuticular hydrocarbon profiles of females were determined, with emphasis on (Z)-9-tricosene (muscalure). Overall, only 55% of mating females had detectable amounts (> 4 etag per fly) of (Z)-9-tricosene. Of the females that had detectable (Z)-9-tricosene, variation in amount per female was high in all fly populations, and thus was not statistically related to the size or age of the mating female. The proportion of mating females with detectable levels of (Z)-9-tricosene varied by geographic region. Seventy-one, 63, and 27% of females from southern California, Minnesota and Georgia had detectable amounts of (Z)-9-tricosene. Principal components analysis of the eight most abundant hydrocarbons from mating females, by state, revealed state-level distinctiveness of hydrocarbons in house fly populations, which may reflect genetic variation associated with environmental stresses in those geographical zones.

Culicoides bottimeri as a vector of Haemoproteus lophortyx to quail in California, USA.

Arthropod sampling via periodic direct bird examination and regular light trapping was conducted between June 2000 and October 2002 to survey for potential vectors of Haemoproteus lophortyx to nonnative, captive-raised bobwhite quail (Colinus virginianus) in northern California, USA. Examination of individual bobwhite quail (from 5 weeks of age through adult, total n=76) was conducted on several dates during the transmission period (June-October). No ectoparasites, including hippoboscid flies (reported as Haemoproteus vectors to wild quail in early literature), were collected from the birds. Trapping with ultraviolet light suction traps near the quail revealed nine species of biting midges (Culicoides spp.). Of these, 94% were C. bottimeri, which was abundant near the birds, and 65% of collected C. bottimeri were engorged with blood. C. bottimeri adult activity began in late-April, slightly before the onset of disease in the quail. Activity peaked between July and late-September, coincident with maximum reported transmission, and adult activity ceased by early-November. Nonengorged C. bottimeri had a parity rate of 43.6% overall, suggesting excellent survival for biological transmission of a pathogen like H. lophortyx. A controlled study was done injecting a macerated slurry of pooled, nonengorged, wild-caught C. bottimeri into the peritoneum of 1-day-old bobwhite quail hatchlings held in insect-proof containers. Blood smears 13-19 days later confirmed H. lophortyx infection in zero controls but all insect-injected quail. Biting midges, especially C. bottimeri, transmit H. lophortyx to captive quail and probably are the dominant vector to native California quail (Callipepla californica) as well.

Duration of viraemia infectious to Culicoides sonorensis in bluetongue virus-infected cattle and sheep.

The duration of viraemia infectious to Culicoides sonorensis (C. sonorensis) was evaluated in bluetongue virus (BTV)-infected sheep and cattle by feeding laboratory-reared C. sonorensis directly on the skin of ruminants that previously were infected with BTV by insect inoculation. The intervals after infection when infectious BTV and BTV nucleic acids were present in the blood of infected cattle and sheep, respectively, were determined by virus isolation and reverse transcriptase (RT)-nested polymerase chain reaction (PCR) assays. The presence of BTV in vector insects that fed on the BTV-infected cattle and sheep at 7, 21, and 49 days post-infection (p.i.) was also determined by virus isolation and RT-PCR assays. BTV was isolated from the blood of infected cattle for up to 49 days p.i., whereas it was not isolated from the BTV-infected sheep after 11 days p.i. In contrast, BTV nucleic acids were detected in the blood of infected ruminants for 111-222 days p.i. The maximal duration of viraemia that was infectious to C. sonorensis was 21 days p.i. of both cattle and sheep and, with the notable exception of one sheep at 21 days p.i., only ruminants whose blood contained BTV as determined by virus isolation were able to infect C. sonorensis after oral feeding. Data from this and previous studies indicates that viraemia is transient in BTV-infected ruminants, and that the RT-nested PCR assay provides a very sensitive and conservative test for the screening of cattle and sheep for the presence of BTV.

A quantitative survey of Culicoides variipennis (Diptera: Ceratopogonidae) in dairy wastewater ponds in southern California.

Culicoides variipennis (Coquillett) larvae were sampled five times over a 10-wk period in late summer in each of 26 dairy wastewater ponds in western Riverside County, Calif. Slope, degree of cattle access, prevalence of visible manure solids, salinity, chemical oxygen demand, plankton volume, and whether the ponds were pumped for irrigation were determined concurrently. Larvae of C. variipennis were associated positively with plankton volume, degree of animal access, and salinity, and negatively with slope and the pumping of the ponds for irrigation. High densities of larvae were found in shoreline mud in shallow, "evaporation bed" type ponds that were not pumped (means = 106 larvae per 30 ml). In contrast, pumped ponds were deeper, had steeper slopes, and supported lower densities of C. variipennis (means = 52 larvae per 30 ml). The pumped ponds tended to hold water continuously (more stable in time), and to have lower phytoplankton volumes and lower values of chemical oxygen demand. Rapid water level fluctuations in the pumped ponds also may have reduced C. variipennis density.

Flight activity and response to carbon dioxide of Culicoides variipennis sonorensis (Diptera: Ceratopogonidae) in southern California.

Adult Culicoides variipennis sonorensis Wirth & Jones were collected using miniature suction traps during 12 nights in October and November at a southern California dairy. Traps were baited with carbon dioxide at a constant release rate of 300, 1,000, or 3,000 ml/min or with 1.3 kg of dry ice. The release rate of CO2 from the dry ice in the laboratory declined from 1,486 ml/min during the first 2 h to 303 ml/min after 10-12 h. Nulliparous and parous females responded in a similar and positive manner to increasing CO2 release rates. An automatic interval suction trap baited with CO2 was used to assess diel host-seeking periodicity. Peak host-seeking activity occurred 0-3 h after sunset, when the trap collected up to 170 females or 103 males per hour. Average peak time of collection during the dusk period was 45 min after sunset. Although numbers were lower, parity was higher in the middle of the night. A smaller, secondary peak of activity occurred near sunrise.

Response of Culicoides variipennis (Diptera: Ceratopogonidae) to water level fluctuations in experimental dairy wastewater ponds.

Water levels were fluctuated in experimental dairy wastewater ponds in southern California to study the response of Culicoides variipennis (Coquillet). Immatures consistently were found in surface mud at the water's edge in ponds where the water level was stable. In ponds where the water level was fluctuated, immatures did not relocate to the new, lower waterline when the water quickly receded 3 m in horizontal distance. Younger larvae apparently were stranded above the waterline and were killed by heat or desication, or both; pupae and late fourth instars emerged as adults at the old waterline. A larvae were recovered from fairly dry mud up to 19 d after the water level receded. Substantial oviposition occurred at the lower waterline following the water level decrease, and adults began to emerge 14-18 d later. Immatures relocated to the higher waterline 3 h to 2 d after a rise in water level. Densities of this species were slightly, but not significantly, reduced in the fluctuated ponds under this water management regime. Water level fluctuations at 1-wk intervals might be expected to reduce substantially C. variipennis production from these habitats.

Improved rearing methods for Heleidomermis magnapapula (Nematoda: Mermithidae), a larval parasite of Culicoides variipennis sonorensis (Diptera: Ceratopogonidae).

Improvements in rearing techniques have decreased maintenance needs and have increased the parasitism rate of larval Culicoides variipennis sonorensis Wirth & Jones by the mermithid nematode, Heleidomermis magnapapula Poinar & Mullens. In colony pans with 2,500-3,000 larval C. v. sonorensis, H. magnapapula adults emerged from 22.5% of hosts, with an average yield of 159 female H. magnapapula per pan. H. magnapapula females refrigerated for 1 wk showed no decline in preparasite infectivity compared with nonrefrigerated females. Female insemination did not differ with various mating combinations of 1 or 2 females with 1 or 2 males; 37-47% of these pairs failed to produce fertilized eggs. In 9-cm petri dishes, the optimal preparasite/host ratio was 900 infective preparasites (1 female's entire clutch) to 650-700 C. v. sonorensis larvae. Overall, parasite production was more time- and labor-efficient when larvae were parasitized in regular colony pans compared with petri dishes.

Field morphological variation and laboratory hybridization of Culicoides variipennis sonorensis and C. v. occidentalis (Diptera:Ceratopogonidae) in southern California.

Two field populations of Culicoides variipennis (Coquillett) from southern California, C. v. occidentalis Wirth & Jones from the Salton Sea and C. v. sonorensis Wirth & Jones from a dairy wastewater pond in the Chino Basin, were sampled monthly from February to July (6-7 mo). Morphometric analyses of slide-mounted adults reared from field-collected larvae and pupae indicated that females of the 2 forms were indistinguishable. Two of the standard characters, wing length and mandibular teeth, were correlated with seasonal temperature changes. Males of C. v. sonorensis were distinguishable by the presence of spicules on the aedeagus, which were entirely lacking in C. v. occidentalis. Two populations of C. v. occidentalis (Salton Sea and Bolsa Chica Marsh) and a laboratory strain of C. v. sonorensis hybridized successfully in the laboratory and were maintained for 6 generations. Differential hybrid viability (F1) was observed in reciprocal crosses. Males of C. v. occidentalis mated with females of C. v. sonorensis resulted in a lower egg hatch (7.4%) than did the reciprocal cross (75.6%). Hybrid males displayed spicules on the aedeagus (a character of C. v. sonorensis), but the number of spicules was sometimes reduced compared with parental C. v. sonorensis (AA strain). Spicules in a field population of C. v. sonorensis were similar in number to the laboratory C. v. sonorensis-C. v. occidentalis hybrids. Based on successful hybridization, the 2 forms should be considered closely related. The 2 forms are separated ecologically by the nature and distribution of their larval habitats.