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.

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.

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.

Bluetongue.

Summary Bluetongue (BT) is an arthropod-transmitted viral disease of non-African ungulates, principally sheep. The disease results from vascular injury analogous to that of human haemorrhagic viral fevers, with characteristic tissue infarction, haemorrhage, vascular leakage, oedema, and hypovolaemic shock. Importantly, BT is not zoonotic. Bluetongue virus (BTV) infection of ruminants and vector Culicoides midges is endemic throughout many tropical and temperate regions of the world; however, within this global range the virus exists within relatively discrete ecosystems (syn. episystems) where specific constellations of BTV serotypes are spread by different species of biting Culicoides midges. Recently discovered goat-associated BTVs, notably BTV serotype 25 (BTV-25) in central Europe, appear to have distinctive biological properties and an epidemiology that is not reliant on Culicoides midges as vectors for virus transmission. Bluetongue virus infection of ruminants is often subclinical, but outbreaks of severe disease occur regularly at the upper and lower limits of the virus's global range, where infection is distinctly seasonal. There have been recent regional alterations in the global distribution of BTV infection, particularly in Europe. It is proposed that climate change is responsible for these events through its impact on vector midges. However, the role of anthropogenic factors in mediating emergence of BTV into new areas remains poorly defined; for example, it is not clear to what extent anthropogenic factors were responsible for the recent translocation to northern and eastern Europe of live attenuated vaccine viruses and an especially virulent strain of BTV-8 with distinctive properties. Without thorough characterisation of all environmental and anthropogenic drivers of the recent emergence of BT in northern Europe and elsewhere, it is difficult to predict what the future holds in terms of global emergence of BTV infection. Accurate and convenient laboratory tests are available for the sensitive and specific serological and virological diagnosis of BTV infection and confirmation of BT in animals. Prevention and control strategies for BT are largely reactive in nature, and typically are reliant on vaccination of susceptible livestock and restrictions on animal trade and movement.

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.

Effects of two-stage weaning with nose flap devices applied to calves on cow body condition, calf performance, and calf humoral immune response.

The effects of nose flap devices in calves before dam separation on cow BCS, pre- and postseparation calf performance, and humoral immune response were compared with traditional weaning. Primiparous and multiparous Angus and Hereford cows ( = 113) and their Angus, Hereford, and Angus × Hereford calves (179.4 ± 3.92 kg and 161 ± 22.7 d of age) were used. Cow-calf pairs were allocated to 1 of 2 treatments in a completely randomized design: 1) nose flap for 21 d before separation from the dam (NF) or 2) no nose flap for 21 d before separation from the dam (CON). Calves were separated from dams on d 0, and calves were placed in group feed-yard pens for 28 d. A subset ( = 75) of weaned calves were placed into 1 of 8 pens to evaluate DMI. Cow BCS was measured on d -21 and 56, and calves were given modified live vaccinations (d -21 and 1), challenged with ovalbumin (OVA; d 1), and weighed (d -21, 1, 7, 14, 21, and 28). In addition, blood samples were collected (d -21, 1, 14, and 28) to measure primary humoral immune response. Control calves tended to have greater BW on d 14 ( = 0.09) and 21 ( = 0.07) than NF calves, and CON calves had greater ( < 0.05) ADG from d -21 to 1 vs. NF calves. Treatments did not differ ( ≥ 0.27) for postweaning DMI, G:F, or morbidity. Serum neutralization tests for bovine viral diarrhea virus type 1 (BVDV-1) and bovine herpesvirus type 1 (BHV-1) were used to measure humoral response to a viral vaccination. Serum antibody titers to BVDV-1 for CON calves tended ( = 0.08) to be greater on d 1 and were greater ( < 0.05) by d 28 vs. NF calves. By d 28, a greater percentage ( < 0.05) of CON calves seroconverted for BVDV-1 than NF calves (82.1 vs. 66.7%, respectively). Serum antibody titers for BHV-1 were greater ( < 0.05) on d 1 and 28 for CON vs. NF calves. Humoral immune response to OVA during the 28-d postseparation period from the dam was evaluated in a subset ( = 57) of calves. There was no difference ( = 0.92) in OVA-specific IgG between treatments on d 14 or 28 ( = 0.76); however, OVA-specific IgM was greater ( < 0.05) in CON vs. NF calves on d 28. Results indicate that nose flap devices did not influence feed intake, feed efficiency, or morbidity during the initial postseparation period from the dam. However, preweaning ADG, serum BVDV-1 and BHV-1 titers, and humoral immune response to OVA were decreased in calves that received the nose flap treatment.

Colostral transmission of bluetongue virus nucleic acid among newborn dairy calves in California.

There have been substantial recent changes in the global distribution and nature of bluetongue virus (BTV) infection of ungulates, perhaps as a result of climate change. To evaluate the epidemiology of BTV infection in California, an area historically endemic for the virus, we monitored newborn dairy calves at different sites for 1 year for the presence of BTV RNA and virus-specific antibodies. The data confirm both localized, vector-mediated, seasonal transmission of BTV as well as dissemination of BTV and/or viral nucleic acid to newborn calves following ingestion of colostrum.

Distribution accuracy of a decentralized unit dose system.

Unit dose medication carts in a 500-bed university hospital were monitored for accuracy and completeness after delivery to the nursing station. The contents of the cart were compared with the nurse's patient medication record. Discrepancies were recorded for evaluation. All medication cart distribution errors found were analyzed to identify the source and were tabulated to determine error rate. Three major categories of errors were discovered: pharmacy technician errors not corrected by the pharmacist, errors associated with nurse's patient medication records, and errors resulting from lost orders.

Is ambulance transport time with lights and siren faster than that without?

STUDY OBJECTIVE: To determine whether ambulance transport time from the scene to the emergency department is faster with warning lights and siren than that without. DESIGN: In a convenience sample, transport times and routes of ambulances using lights and sirens were recorded by an observer. The time also was recorded by a paramedic who drove an ambulance without lights and siren over identical routes during simulated transports at the same time of day and on the same day of the week as the corresponding lights-and-siren transport. SETTING: An emergency medical service system in a city with a population of 46,000. PARTICIPANTS: Emergency medical technicians and paramedics. RESULTS: Fifty transport times with lights and siren averaged 43.5 seconds faster than the transport times without lights and siren [t = 4.21, P = .0001]. CONCLUSION: In this setting, the 43.5-second mean time savings does not warrant the use of lights and siren during ambulance transport, except in rare situations or clinical circumstances.