Pathogen detection and disease diagnosis in wildlife: challenges and opportunities.

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.

A review of guidelines for evaluating a minor modification to a validated assay.

Any modification to a validated assay must be evaluated in terms of the impact on the assay's performance characteristics and whether the assay remains fit for the intended purpose. The comparison is referred to as a 'method comparison', 'method comparability', 'method change', or 'comparative validation'. This review presents recommendations and examples of studies found in the current literature as a means of assessing minor modifications. In addition, the authors discuss common statistical approaches used for these comparisons.

Toute modification apportée à un essai validé doit être évaluée afin de mesurer l'impact de cette modification sur les paramètres de performances de l'essai et déterminer si l'aptitude à l'emploi qui lui a été assigné demeure valable suite à la modification en question. Cette comparaison est désignée sous les termes de « comparaison de méthodes d'essai ¼, « comparabilité de méthodes ¼, « changement de méthode d'essai ¼ ou « validation comparative ¼. Les auteurs font part de leurs recommandations et donnent des exemples d'études émanant de la littérature récente concernant l'évaluation de modifications mineures. En outre, ils examinent les approches statistiques couramment utilisées pour ces comparaisons.

Toda modificación que se introduzca en un ensayo validado debe ser objeto de evaluación para determinar la influencia del cambio en las características de funcionamiento del ensayo y saber si este sigue estando adaptado a su función. Para referirse a la comparación, los autores emplean expresiones como 'comparación de métodos', 'comparabilidad de métodos', 'cambio de método' o 'validación comparativa'. Los autores presentan aquí recomendaciones y ejemplos de estudios extraídos de la bibliografía actual como medio de evaluar modificaciones de importancia menor. Además, los autores examinan las lógicas estadísticas comunes utilizadas para estas comparaciones.

Bovine herpes virus type-4 infection among postpartum dairy cows in California: risk factors and phylogenetic analysis.

The aims of this study were to determine the prevalence and associated risk factors of bovine herpes virus type-4 (BoHV-4) infection and describe the genetic characteristics and predominant genotypes of the virus in Yolo and Tulare counties, California. A cross-sectional study involving multi-stage sampling technique was used. One hundred and forty-eight post-partum cows were enrolled from 11 dairy farms. Uterine/vaginal samples were collected and tested for BoHV-4 and other co-infecting viruses using real-time PCR. Data were analysed using multilevel logistic mixed-effect model. Phylogenetic analysis of 10 BoHV-4 isolates was conducted by targeting three open reading frames (ORF3, ORF8 and ORF 22) of the viral genome. The prevalence of BoHV-4 infection was 22.3% (33/148), while post-partum metritis was 33.8% (48/142). Strong association was found between BoHV-4 infection and lactation number, lactation stage and post-partum metritis. The odds of being positive for BoHV-4 infection were 6.47 times (95% CI 1.17-35.92; P 240 days). Cows with post-partum metritis were 4.51 times (95% CI 1.27-16.02; P < 0.05) more likely to test positive for BoHV-4 infection compared with those without post-partum metritis. Phylogenetic analysis of BoHV-4 based on sequencing of glycoprotein and thymidine kinase (TK) genes revealed genetic variability of the virus with glycoprotein B genotype 1 and TK genotype 2 as being dominant genotypes. The reported high genetic variability of BoHV-4 indicates the possibility of co-infection with multiple genotypes.

A review of the use of human papilloma virus (HPV) in cervical screening.

Using key words online databases were searched to identify relevant publications to review the use of Human papilloma virus (HPV) in cervical screening. The mode of cervical screening in the UK has been decided but implementation plans have yet to be announced. The protracted uncertainty surrounding the initial announcement to move to HPV primary screening together with the lack of a national steer has resulted in a flight of staff which threatens the provision of the current and future service. The transition will be a challenging time but analysis of data from more than 176,000 women has shown clear evidence of a reduction in the incidence of cancer where HPV testing is used. There will however, be a population of women who are cytologically negative but high-risk HPV positive and the management of these women will be key to maximising the benefits of HPV primary screening. As cervical cytology becomes increasingly rare its effectiveness and role in cervical screening will come under scrutiny and we must ensure the specificity of reporting is maintained in order for it to survive.

Diagnostic exercise: High mortality in a flock of chukar partridge chicks (Alectoris chukar) in California.

Mortality of 20% of a flock of 1000 chukar partridge chicks occurred over a 6-week period in Northern California from August to September 2012. Affected birds were 2 to 42 days old and died without premonitory clinical signs or after showing ruffled feathers and anorexia for 24 to 72 hours. Three carcasses were submitted for necropsy, 2 birds had hemorrhagic tracheitis grossly, and all 3 had lymphoplasmacytic and histiocytic myocarditis with myocardial necrosis microscopically. The differential diagnoses and the diagnostic workup to achieve a final diagnosis are discussed. The detection of 2 zoonotic agents in these birds makes this an interesting case from a public health perspective.

Necrotizing Enteritis and Hyperammonemic Encephalopathy Associated With Equine Coronavirus Infection in Equids.

Equine coronavirus (ECoV) is a Betacoronavirus recently associated clinically and epidemiologically with emerging outbreaks of pyrogenic, enteric, and/or neurologic disease in horses in the United States, Japan, and Europe. We describe the pathologic, immunohistochemical, ultrastructural, and molecular findings in 2 horses and 1 donkey that succumbed to natural infection with ECoV. One horse and the donkey (case Nos. 1, 3) had severe diffuse necrotizing enteritis with marked villous attenuation, epithelial cell necrosis at the tips of the villi, neutrophilic and fibrinous extravasation into the small intestinal lumen (pseudomembrane formation), as well as crypt necrosis, microthrombosis, and hemorrhage. The other horse (case No. 2) had hyperammonemic encephalopathy with Alzheimer type II astrocytosis throughout the cerebral cortex. ECoV was detected by quantitative polymerase chain reaction in small intestinal tissue, contents, and/or feces, and coronavirus antigen was detected by immunohistochemistry in the small intestine in all cases. Coronavirus-like particles characterized by spherical, moderately electron lucent, enveloped virions with distinct peplomer-like structures projecting from the surface were detected by negatively stained transmission electron microscopy in small intestine in case No. 1, and transmission electron microscopy of fixed small intestinal tissue from the same case revealed similar 85- to 100-nm intracytoplasmic particles located in vacuoles and free in the cytoplasm of unidentified (presumably epithelial) cells. Sequence comparison showed 97.9% to 99.0% sequence identity with the ECoV-NC99 and Tokachi09 strains. All together, these results indicate that ECoV is associated with necrotizing enteritis and hyperammonemic encephalopathy in equids.

Development of a technical external quality assurance scheme in non-gynaecological cytology in UK.

Technical external quality assurance (EQA) schemes are well established for histopathology and cervical cytology but, to date, sadly lacking for diagnostic cytology (DC). This timely review redresses the balance by describing the development and evaluation of a technical EQA scheme for DC available to the UK, Europe and beyond.

Diagnostic Detection of H7N3 Low Pathogenicity Avian Influenza in a Commercial Game Bird Flock.

Low pathogenicity avian influenza (LPAI) H7N3 was diagnosed in a flock of commercially raised Japanese quail (Coturnix japonica) breeder hens. The birds were submitted with an initial complaint of a drop in egg production and watery droppings. Initial PCR testing of the flock for avian influenza (AI) was negative; however, mortality started increasing drastically, prompting further examination and the submission of more birds to the diagnostic laboratory. On antemortem examination, the birds appeared lethargic, with ruffled feathers, and had labored breathing. Gross examination revealed poor body condition, moderate dehydration, splenomegaly, ovarian regression, and airsacculitis. Several hens produced thin-shelled and shell-less eggs. Microscopically, lymphoplasmacytic encephalitis with neuronal cell necrosis, fibrinoheterophilic pneumonia, and splenic lymphoid depletion with amyloid deposition were seen. AI virus was detected in a follow-up submission and characterized as LPAI H7N3. Quail are a species of interest in the pathobiology of avian influenza viruses, underlined by the ability to serve as an amplification host.

Reporte de caso- Detección diagnóstica de influenza aviar de baja patogenicidad H7N3 en una parvada comercial de aves de caza. Se diagnosticó influenza aviar de baja patogenicidad (LPAI) H7N3 en una parvada de gallinas reproductoras de codornices japonesas criadas comercialmente. Las aves se recibieron con una historia inicial de caída en la producción de huevo y excrementos acuosos. La prueba inicial de PCR para influenza aviar de la parvada fue negativa; sin embargo, la mortalidad comenzó a aumentar drásticamente, lo que generó posteriores evaluaciones y el envío de más aves al laboratorio de diagnóstico. En el examen ante mortem, las aves estaban letárgicas, con las plumas erizadas y tenían dificultad para respirar. El examen macroscópico reveló mala condición corporal, deshidratación moderada, esplenomegalia, regresión ovárica y aerosaculitis. Varias gallinas produjeron huevos con cascarón delgado o sin cascarón. Microscópicamente, se observaron encefalitis linfoplasmocítica con necrosis de células neuronales, neumonía fibrinoheterófila y depleción linfoide esplénica con depósito de amiloide. El virus de influenza aviar se detectó en un caso de seguimiento y se caracterizó como de baja patogenicidad subtipo H7N3. Las codornices son una especie de interés en la patobiología de los virus de la influenza aviar, subrayada por la capacidad de servir como huésped de amplificación.

Avian Reoviruses of the Same Genotype Induce Different Pathology in Chickens.

The emergence of avian reovirus variant strains has caused negative effects in the poultry industry worldwide. Regardless of the efforts in molecular characterization and classification of these variants, information about the pathogenicity, transmissibility, and immunosuppression in chickens is limited. The genomes of two variant strains (A and B) and a classic S1133 strain (C) belonging to the same sigma C genotype 1 were compared. Additionally, these strains were used in a challenge experiment to evaluate inoculated and indirectly exposed specific-pathogen-free chickens. The whole-genome sequence analysis of the three strains revealed nucleotide identity differences in the L3, M2, and S1 genes. Strains A and B also showed homology differences in the S4 gene, despite having high homologies in all other genes. The in vivo challenge experiments showed that, whereas variant A induced high viral loads in tendons, hearts, and duodena of inoculated chickens, variant B induced high viral loads in indirectly exposed chickens. Likewise, histopathology reflected differences in the pathologic effects induced by these strains. For instance, the B and C strains induced more severe microscopic lesions compared with the A strain. Lymphoid depletion was more severe in bursas than in thymi, and inoculated birds were more affected than exposed birds. In conclusion, different pathologic outcomes in chickens were observed depending on the strain and transmission route. This study provides insights onto the relationship between pathogenicity and genomic composition of avian reoviruses.

Reovirus aviares del mismo genotipo inducen diferentes patologías en pollos. La aparición de cepas variantes del reovirus aviar ha causado efectos negativos en la industria avícola en todo el mundo. Independientemente de los esfuerzos en la caracterización molecular y clasificación de estas variantes, la información sobre la patogenicidad, transmisibilidad e inmunodepresión en pollos es limitada. Se compararon los genomas de dos cepas variantes (A y B) y una cepa S1133 clásica (C) perteneciente al mismo genotipo 1 del gene sigma C. Además, estas cepas se utilizaron en un experimento de desafío para evaluar pollos libres de patógenos específicos inoculados y expuestos indirectamente. El análisis de la secuencia del genoma completo de las tres cepas reveló diferencias de identidad de nucleótidos en los genes L3, M2 y S1. Las cepas A y B también mostraron diferencias de homología en el gene S4, a pesar de tener altas similitudes en todos los demás genes. Los experimentos de exposición in vivo mostraron que, mientras que la variante A inducía altas cargas virales en tendones, corazones y duodeno en los pollos inoculados, la variante B inducía altas cargas virales en pollos expuestos indirectamente. Asimismo, la histopatología reflejó diferencias en los efectos patológicos inducidos por estas cepas. Por ejemplo, las cepas B y C indujeron lesiones microscópicas más graves en comparación con la cepa A. La despoblación linfoide fue más severa en las bolsas que en el timo, y las aves inoculadas resultaron más afectadas que las expuestas. En conclusión, se observaron diferentes resultados patológicos en pollos según la cepa y la vía de transmisión. Este estudio proporciona información sobre la relación entre la patogenicidad y la composición genómica de los reovirus aviares.

Infectious Bronchitis Virus Surveillance in Broilers in California (2012-20).

Infectious bronchitis virus (IBV) causes severe economic losses among chicken flocks worldwide. Although IBV molecular surveillance has been conducted in California broilers, seasonal and spatial-temporal trends in IBV prevalence are poorly defined. The goals of this study were to evaluate seasonal and spatial-temporal trends in IBV prevalence and to determine the predominant IBV genotypes obtained over the last 8 yr from a broiler company located in the California Central Valley. In total, 3439 broilers with a suspicion of IBV infection were submitted to the California Animal Health and Food Safety laboratories between January 2012 and February 2020. Swabs from tracheas, kidneys, and cecal tonsils from each submission were independently pooled and screened for IBV using reverse transcriptase quantitative PCR (RT-qPCR). Positive samples were submitted for virus isolation. Viral isolates were subject to a conventional RT-PCR targeting the S1 gene hypervariable region. Positive samples from this RT-PCR were sequenced, and phylogenetic analyses were performed. In total, 1243 pooled swab samples were positive for IBV. Positive results were more frequently detected in fall and winter months compared to spring. Spatial analyses revealed an IBV hot spot in the vicinity of Livingston, and two areas with a low prevalence (i.e., cold spots) around Riverdale. The IBV spatial-temporal distribution identified three significant clusters: one hot spot around Turlock from 2015 to 2016, a second hot spot around Merced from 2012 to 2016, and a cold spot around Fresno from 2017 to 2020. Predominant genotypes changed over time from IBV Cal 99, which was predominant between 2012 and 2014, to IBV 3099 in 2019. Vaccination efforts were initiated in 2018, and as a result, we detected an emerging variant with 92% similarity to CA 3099 in 2020. This work highlights the importance of ongoing surveillance in IBV prevention programs. Surveillance strategies are necessary to monitor trends in diseases such as infectious bronchitis, and the tools used for surveillance need to be sensitive enough to detect new variants and identify spatial-temporal trends.

Vigilancia del virus de la bronquitis infecciosa en pollos de engorde en California (2012­2020). El virus de la bronquitis infecciosa (IBV) causa graves pérdidas económicas entre las parvadas de pollos en todo el mundo. Aunque la vigilancia molecular del virus de la bronquitis infecciosa se ha realizado en pollos de engorde en el estado de California, las tendencias estacionales y espacio-temporales sobre la prevalencia de este virus están mal definidas. Los objetivos de este estudio fueron evaluar las tendencias estacionales y espacio-temporales sobre la prevalencia del virus de la bronquitis infecciosa y determinar los genotipos predominantes de este virus obtenidos durante los últimos ocho años de una empresa de pollos de engorde ubicada en el Valle Central de California. En total, 3439 pollos de engorde con sospecha de infección por el virus de la bronquitis infecciosa se enviaron a los laboratorios de Salud Animal y Seguridad Alimentaria del estado de California entre enero del 2012 y febrero del 2020. Los hisopos de tráqueas, riñones y tonsilas cecales de cada caso se combinaron de forma independiente y se examinaron para detectar al virus de la bronquitis utilizando transcripción reversa y un método cuantitativo de PCR (RT-qPCR). Se enviaron muestras positivas para aislamiento del virus. Los aislados virales se sometieron a un método convencional de RT-PCR dirigido a la región hipervariable del gene S1. Se secuenciaron muestras positivas mediante la prueba RT-PCR y se realizaron análisis filogenéticos. Un total de 1243 muestras combinadas de hisopos dieron positivo para el virus de la bronquitis infecciosa. Los resultados positivos se detectaron con mayor frecuencia en los meses de otoño e invierno en comparación con la primavera. Los análisis espaciales revelaron un punto activo para el virus de la bronquitis infecciosa en las cercanías de Livingston y dos áreas con una baja prevalencia (es decir, puntos fríos) alrededor de Riverdale. La distribución espacio-temporal del virus de la bronquitis identificó tres grupos importantes: un punto activo alrededor de Turlock entre los años 2015 a 2016, un segundo punto activo alrededor de Merced entre los años 2012 a 2016 y un punto frío alrededor de Fresno entre los años 2017 a 2020. Los genotipos predominantes cambiaron con el tiempo, consideraron el subtipo IBV Cal 99, que fue predominante entre 2012 y 2014, a el tipo IBV 3099 en 2019. Los esfuerzos de vacunación se iniciaron en el 2018, y como resultado, detectamos una variante emergente con un 92% de similitud con el virus CA 3099 en 2020. Este trabajo destaca la importancia de la vigilancia continua en los programas de prevención para la bronquitis infecciosa. Las estrategias de vigilancia son necesarias para monitorear las tendencias en enfermedades como la bronquitis infecciosa, y las herramientas utilizadas para la vigilancia deben ser lo suficientemente sensibles como para detectar nuevas variantes e identificar tendencias espacio-temporales.

Equine herpesvirus-1 genotype did not significantly affect clinical signs and disease outcome in 65 horses diagnosed with equine herpesvirus-1 myeloencephalopathy.

The objective of this study was to determine if the genotype of equine herpesvirus-1 (EHV-1) impacted clinical disease and outcome of horses with laboratory confirmed equine herpesvirus myeloencephalopathy (EHM). Medical records from 65 horses diagnosed with EHM from 2011 to 2019 were reviewed for signalment, presence and severity of clinical signs (lethargy, fever, ataxia, urinary incontinence) and outcome. Horses were further grouped based on the EHV-1 genotype into neuropathic (D752) or non-neuropathic (N752) EHV-1 infection. Between the two EHV-1 genotype groups, age and sex distributions were similar, although breed distribution was different (Quarter Horses and Saddlebreds were overrepresented and Warmbloods were underrepresented in the EHV-1 D752 group compared to the EHV-1 N752 group; P = 0.009). Lethargy, fever, ataxia and outcome were not significantly different between the two EHV-1 genotype groups (P > 0.05). However, urinary incontinence was significantly more frequently reported in horses infected with the D752 genotype of EHV-1 (P=0.04). Contrary to previous studies, the present study showed no difference in frequency of genotype (D752 or N752) among 65 horses with EHM and, with the exception of urinary incontinence, no difference in clinical disease or outcome related to the EHV-1 genotype.

Genotypic Characterization of Emerging Avian Reovirus Genetic Variants in California.

This study focuses on virus isolation of avian reoviruses from a tenosynovitis outbreak between September 2015 and June 2018, the molecular characterization of selected isolates based on partial S1 gene sequences, and the full genome characterization of seven isolates. A total of 265 reoviruses were detected and isolated, 83.3% from tendons and joints, 12.3% from the heart and 3.7% from intestines. Eighty five out of the 150 (56.6%) selected viruses for sequencing and characterization were successfully detected, amplified and sequenced. The characterized reoviruses grouped in six distinct genotypic clusters (GC1 to GC6). The most represented clusters were GC1 (51.8%) and GC6 (24.7%), followed by GC2 (12.9%) and GC4 (7.2%), and less frequent GC5 (2.4%) and GC3 (1.2%). A shift on cluster representation throughout time occurred. A reduction of GC1 and an increase of GC6 classified strains was noticed. The highest homologies to S1133 reovirus strain were detected in GC1 (~77%) while GC2 to GC6 homologies ranged between 58.5 and 54.1%. Over time these homologies have been maintained. Seven selected isolates were full genome sequenced. Results indicated that the L3, S1 and M2 genes, coding for proteins located in the virus capsid accounted for most of the variability of these viruses. The information generated in the present study helps the understanding of the epidemiology of reoviruses in California. In addition, provides insights on how other genes that are not commonly studied add variability to the reovirus genome.

Allocating external financing for health: a discrete choice experiment of stakeholder preferences.

Most donors of external financing for health use allocation policies to determine which countries are eligible to receive financial support and how much support each should receive. Currently, most of these policies place a great deal of weight on income per capita as a determinant of aid allocation but there is increasing interest in putting more weight on other country characteristics in the design of such policies. It is unclear, however, how much weight should be placed on other country characteristics. Using an online discrete choice experiment designed to elicit preferences over country characteristics to guide decisions about the allocation of external financing for health, we find that stakeholders assign a great deal of importance to health inequalities and the burden of disease but put very little weight on income per capita. We also find considerable variation in preferences across stakeholders, with people from low- and middle-income countries putting more weight on the burden of disease and people from high-income countries putting more weight on health inequalities. These findings suggest that stakeholders put more weight on burden of disease and health inequalities than on income per capita in evaluating which countries should received external financing for health and that that people living in aid recipient may have different preferences than people living in donor countries. Donors may wish to take these differences in preferences in mind if they are reconsidering their aid allocation policies.

Persistence of Highly Pathogenic and Low Pathogenic Avian Influenza Viruses in Footbaths and Poultry Manure.

A questionnaire was designed in order to gather information about bedding material and footbath preparation and maintenance in different productive units across the state of California.This information was used to plan two experiments. In the first experiment, we tested the effectiveness of footbaths in inactivating highly pathogenic (HP) and low pathogenic (LP) avian influenza viruses (AIVs) on rubber boots. Surprisingly, quaternary ammonia- and quaternary ammonia + glutaraldehyde-based footbaths were not able to eliminate live HPAIV (H5N8) and LPAIV (H6N2) particles on boots, while a chlorine-based granulated disinfectant was able to destroy the virus at contact. These results demonstrated the potential of AIV, particularly the HPAIV isolate, to persist even if exposed to disinfecting footbaths, and suggest that footbaths, as a single tool, are not capable of preventing pathogen introduction into commercial flocks. In the second experiment, we investigated the persistence of HPAIV (H5N8) and LPAIV (H6N2) in bedding material and feces obtained from turkey, broiler, and egg-layer commercial productive units. Samples were collected at different times after spiking the bedding materials and feces. Results showed that HPAIV (H5N8) was more persistent than LPAIV (H6N2) in layer feces and bedding material obtained from commercial broilers and turkeys. Live HPAIV particles persisted 96 hr, the last time point measured, in layer feces and less than 60 hr in broiler and turkey bedding. In contrast, LPAIV persisted less than 24 hr after being spiked in all the different substrates. Further research in biosecurity practices such as footbath preparation and maintenance and better understanding of the mechanism of the increased persistence of AIV is warranted in order to identify effective litter treatments that destroy live virus in bedding material.

Agreement Among 4 Sampling Methods to Identify Respiratory Pathogens in Dairy Calves with Acute Bovine Respiratory Disease.

BACKGROUND: Four sampling techniques commonly are used for antemortem identification of pathogens from cattle with bovine respiratory disease (BRD): the nasal swab (NS), guarded nasopharyngeal swab (NPS), bronchoalveolar lavage (BAL), and transtracheal wash (TTW). Agreement among these methods has not been well characterized. OBJECTIVE: To evaluate agreement among TTW and NS, NPS, or BAL for identification of viral and bacterial pathogens in dairy calves with BRD. ANIMALS: One hundred dairy calves with naturally acquired BRD. METHODS: Calves were sampled by all 4 methods. Viral agents were identified by real-time RT-PCR, bacteria were identified by aerobic culture, and Mycoplasma bovis (M. bovis) isolates were speciated by PCR. Agreement among TTW and NS, NPS, or BAL was evaluated by calculating the kappa statistic and percent positive agreement. McNemar's exact test was used to compare the proportions of positive results. RESULTS: Agreement among TTW and NS, TTW and NPS, and TTW and BAL, was very good for identification of P. multocida, M. haemolytica, and M. bovis. For bovine respiratory syncytial virus (BRSV), agreement with TTW was moderate for NS, good for NPS, and very good for BAL. For bovine coronavirus (BCV), agreement with TTW was moderate for NS and NPS, and good for BAL. McNemar's test was significant only for BCV, indicating that for this pathogen the proportion of positive results from NS and NPS could not be considered comparable to TTW. CONCLUSIONS AND CLINICAL IMPORTANCE: This study provides guidance for veterinarians selecting diagnostic tests for antemortem identification of pathogens associated with BRD.

Mechanisms of human tumor metastasis studied in patients with peritoneovenous shunts.

The technique of peritoneovenous shunting for the alleviation of abdominal pain and distension in malignant ascites due to inoperable cancer, returns the fluid to the circulation via a one-way, valved, anastomosis between the peritoneum and the jugular vein. Surprisingly, although the patients treated with this technique receive direct infusions of malignant tumor cells into the blood, this study of 29 patients, 15 of whom came to autopsy, shows that they did not all develop metastases, some being completely free of such lesions despite long survival. Even when metastases do form, they are small and clinically asymptomatic, and the technique is therefore not hazardous. In some patients, inert tumor cells identifiable by natural markers were recognized in the tissues, but no growing metastases were observed. In others, the distribution of secondary deposits was unexpected in that metastases did not form in the organ containing the first capillary bed encountered, although hematogenous metastases had formed in other organs. Despite the fact that various factors such as (a) the small numbers of patients treated with the technique; (b) the sensitive nature of studies on terminally ill patients; and (c) the absence of consistency in the sample population with regard to factors such as length of survival and site of neoplasm, combine to reduce the number of suitable cases for study, the approach has unrivaled power and interest for those seeking to understand mechanisms underlying tumor metastasis in humans.

General practitioner notes as a source of information for case-control studies in young women. UK National Case-Control Study Group.

STUDY OBJECTIVE: The UK National Case-Control Study was carried out to investigate the relationship between oral contraceptive use and breast cancer risk. This study investigates whether general practitioner notes could be used as the sole data source for epidemiological studies of young women and what the effect would be on non-response and recall bias. DESIGN: Case-control study with data on gynaecological, obstetric, and contraceptive history collected at interview and from general practitioners' notes. Information from these two sources was compared. SETTING: This was a population-based study. PARTICIPANTS: Altogether 755 women with breast cancer aged under 36 years at diagnosis, each with an age-matched control, participated in the study. Response rates at interview were 72% and 89% for cases and controls but GP data were available for 90% of the 1049 case and first-selected control pairs. MAIN RESULTS: There was generally good agreement between the two data sources with respect to obstetric history and gynaecological procedures (hysterectomy, oophorectomy, and tubal ligation). The use of intra-uterine devices, or diaphragm, and partner's vasectomy were not reliably recorded in the GP's notes. The overall results of the UK study would have been qualitatively the same with respect to the relationship between oral contraceptive use and breast cancer risk if GP notes only had been used, in spite of the fact that only about half of all oral contraceptive usage was recorded in the notes. Response rates would have been higher, recall bias eliminated, and the cost of the study halved. CONCLUSIONS: When planning case-control studies in young women, the possibility of using GP notes as the primary data source should be considered. Lack of data on potential confounding factors is a possible drawback to such use. The practice of destroying GP's notes shortly after the death of patients seriously restricts the possibility of using these notes when studying rapidly fatal conditions.

Effect on attendance at breast cancer screening of adding a self administered questionnaire to the usual invitation to breast screening in southern England.

STUDY OBJECTIVE: The primary aim of the research described in this paper was to ascertain whether inclusion of a self administered questionnaire with the usual invitation to routine breast screening affected screening uptake. Secondary aims included establishing appropriate questionnaire distribution and collection methods within the framework of the National Health Service Breast Screening Programme and optimisation of questionnaire design. DESIGN: Randomised study. SETTING: Oxfordshire and West of London Breast Screening Units. PARTICIPANTS/METHODS: 6400 women invited for routine screening mammography were individually randomised to receive either the usual breast screening invitation alone, or to receive the usual invitation accompanied by a self administered questionnaire, to be returned at the time of screening. Participants were then followed up and attendance rates at screening were compared between groups. MAIN RESULTS: Screening attendance rates were similar in women who did and did not receive a questionnaire (71% in each group). Of those who were sent a questionnaire and attended for screening, 77% returned a completed questionnaire. Screening uptake was not affected by the way in which the questionnaire was returned or by whether or not personal details and signed permission for follow up were requested. CONCLUSIONS: The inclusion of a self administered questionnaire accompanying the invitation to breast screening did not adversely affect screening uptake. A high proportion of women completed the questionnaire.

Predictors of outcome of mammography in the National Health Service Breast Screening Programme.

BACKGROUND: Little is known about the factors influencing the risk of recall for assessment, invasive diagnostic procedures, and early rescreening after screening mammography. METHODS: From June 1996 to March 1998 women attending screening at 10 National Health Service Breast Screening Programme (NHSBSP) centres completed a self administered questionnaire and were followed up for their screening outcome. RESULTS: 1969 (3.3%) out of 60 443 women aged 50-64 who had never used hormone replacement therapy (HRT) were recalled for assessment but were not diagnosed with breast cancer (defined here as false positive recall). After adjustment for the variation between centres, false positive recall was decreased significantly among women who were likely to have had a previous NHSBSP mammogram (odds ratio (OR) 0.49, 95% confidence interval (95% CI) 0.38 to 0.63 for likely versus unlikely), who were postmenopausal (OR 0.65, 95% CI 0.56 to 0.76 for postmenopausal v premenopausal) and increased significantly for women reporting previous breast surgery (OR 1.64, 95% CI 1.42 to 1.89). Although false positive recall decreased significantly with parity and increasing body mass index, these effects were not large and no significant variation was found with age, education, family history of breast cancer, oral contraceptive use, sterilisation, exercise, smoking, or alcohol consumption. Altogether 655 (1.1%) women had an invasive diagnostic procedure; no personal characteristics were predictive of this outcome, 286(0.5%) were referred for early rescreening, and this was increased significantly by nulliparity and a family history of breast cancer. INTERPRETATION: Premenopausal women, those without a previous NHSBSP mammogram, and women with previous breast surgery have an increased risk of false positive recall by the NHSBSP.