Analysis of geographic location and pathways for influenza A virus infection of commercial upland game bird and conventional poultry farms in the United States of America.

BACKGROUND: Avian influenza (AI) is an infectious viral disease that affects several species and has zoonotic potential. Due to its associated health and economic repercussions, minimizing AI outbreaks is important. However, most control measures are generic and mostly target pathways important for the conventional poultry farms producing chickens, turkeys, and eggs and may not target other pathways that may be specific to the upland game bird sector. The goal of this study is to provide evidence to support the development of novel strategies for sector-specific AI control by comparing and contrasting practices and potential pathways for spread in upland game bird farms with those for conventional poultry farms in the United States. Farm practices and processes, seasonality of activities, geographic location and inter-farm distance were analyzed across the sectors. All the identified differences were framed and discussed in the context of their associated pathways for virus introduction into the farm and subsequent between-farm spread. RESULTS: Differences stemming from production systems and seasonality, inter-farm distance and farm densities were evident and these could influence both fomite-mediated and local-area spread risks. Upland game bird farms operate under a single, independent owner rather than being contracted with or owned by a company with other farms as is the case with conventional poultry. The seasonal marketing of upland game birds, largely driven by hunting seasons, implies that movements are seasonal and customer-vendor dynamics vary between industry groups. Farm location analysis revealed that, on average, an upland game bird premises was 15.42 km away from the nearest neighboring premises with birds compared to 3.74 km for turkey premises. Compared to turkey premises, the average poultry farm density in a radius of 10 km of an upland game bird premises was less than a half, and turkey premises were 3.8 times (43.5% compared with 11.5%) more likely to fall within a control area during the 2015 Minnesota outbreak. CONCLUSIONS: We conclude that the existing differences in the seasonality of production, isolated geographic location and epidemiological seclusion of farms influence AI spread dynamics and therefore disease control measures should be informed by these and other factors to achieve success.

Quantifying the effect of swab pool size on the detection of influenza A viruses in broiler chickens and its implications for surveillance.

BACKGROUND: Timely diagnosis of influenza A virus infections is critical for outbreak control. Due to their rapidity and other logistical advantages, lateral flow immunoassays can support influenza A virus surveillance programs and here, their field performance was proactively assessed. The performance of real-time polymerase chain reaction and two lateral flow immunoassay kits (FluDETECT and VetScan) in detecting low pathogenicity influenza A virus in oropharyngeal swab samples from experimentally inoculated broiler chickens was evaluated and at a flock-level, different testing scenarios were analyzed. RESULTS: For real-time polymerase chain reaction positive individual-swabs, FluDETECT respectively detected 37% and 58% for the H5 and H7 LPAIV compared to 28% and 42% for VetScan. The mean virus titer in H7 samples was higher than for H5 samples. For real-time polymerase chain reaction positive pooled swabs (containing one positive), detections by FluDETECT were significantly higher in the combined 5- and 6-swab samples compared to 11-swab samples. FluDETECT detected 58%, 55.1% and 44.9% for the H7 subtype and 28.3%, 34.0% and 24.6% for the H5 in pools of 5, 6 and 11 respectively. In our testing scenario analysis, at low flock-level LPAIV infection prevalence, testing pools of 11 detected slightly more infections while at higher prevalence, testing pools of 5 or 6 performed better. For highly pathogenic avian influenza virus, testing pools of 11 (versus 5 or 6) detected up to 5% more infections under the assumption of similar sensitivity across pools and detected less by 3% when its sensitivity was assumed to be lower. CONCLUSIONS: Much as pooling a bigger number of swab samples increases the chances of having a positive swab included in the sample to be tested, this study's outcomes indicate that this practice may actually reduce the chances of detecting the virus since it may result into lowering the virus titer of the pooled sample. Further analysis on whether having more than one positive swab in a pooled sample would result in increased sensitivity for low pathogenicity avian influenza virus is needed.

Historical and Recent Cases of H3 Influenza A Virus in Turkeys in Minnesota.

Subtype H3 influenza A viruses (IAVs) are abundant in wild waterfowl and also infect humans, pigs, horses, dogs, and seals. In Minnesota, turkeys are important and frequent hosts of IAV from wild waterfowl and from pigs. Over 48 yr of surveillance history, 11 hemagglutinin (HA) subtypes of IAV from waterfowl, as well as two HA subtypes from swine, H1 and H3, have infected turkeys in Minnesota. However, there have only been two cases of avian-origin H3 IAV infections in turkeys during this 48-yr period. The first avian-origin IAV infection was detected in seven breeder and commercial flocks in 1982 and was caused by a mixed H3H4/N2 infection. In 2013, an avian-origin H3H9/N2 outbreak occurred in five flocks of turkeys between 15 and 56 wk of age. Phylogenetic analysis of the HA gene segment from the 2013 isolate indicated that the virus was related to a wild bird lineage H3 IAV. A meta-analysis of historical H3 infections in domesticated poultry demonstrated that avian-origin H3 infections have occurred in chickens and ducks but were rare in turkeys. H9N2 virus was subsequently selected during the egg cultivation of the 2013 H3H9/N2 mixed virus. A growth curve analysis suggested that passage 3 of A/Turkey/Minnesota/13-20710-2/2013(mixed) had a slightly lower replication rate than a similar avian-origin H3N2. The challenge studies indicated that the infectious dose of avian-origin H3N2 for turkey poults was greater than 10(6) 50% egg infective dose. Considered together, these data suggest that avian-origin H3 introductions to turkeys are rare events.

Quantitative Estimation of the Number of Contaminated Hatching Eggs Released from an Infected, Undetected Turkey Breeder Hen Flock During a Highly Pathogenic Avian Influenza Outbreak.

The regulatory response to an outbreak of highly pathogenic avian influenza (HPAI) in the United States may involve quarantine and stop movement orders that have the potential to disrupt continuity of operations in the U.S. turkey industry--particularly in the event that an uninfected breeder flock is located within an HPAI Control Area. A group of government-academic-industry leaders developed an approach to minimize the unintended consequences associated with outbreak response, which incorporates HPAI control measures to be implemented prior to moving hatching eggs off of the farm. Quantitative simulation models were used to evaluate the movement of potentially contaminated hatching eggs from a breeder henhouse located in an HPAI Control Area, given that active surveillance testing, elevated biosecurity, and a 2-day on-farm holding period were employed. The risk analysis included scenarios of HPAI viruses differing in characteristics as well as scenarios in which infection resulted from artificial insemination. The mean model-predicted number of internally contaminated hatching eggs released per movement from an HPAI-infected turkey breeder henhouse ranged from 0 to 0.008 under the four scenarios evaluated. The results indicate a 95% chance of no internally contaminated eggs being present per movement from an infected house before detection. Sensitivity analysis indicates that these results are robust to variation in key transmission model parameters within the range of their estimates from available literature. Infectious birds at the time of egg collection are a potential pathway of external contamination for eggs stored and then moved off of the farm; the predicted number of such infectious birds was estimated to be low. To date, there has been no evidence of vertical transmission of HPAI virus or low pathogenic avian influenza virus to day-old poults from hatching eggs originating from infected breeders. The application of risk analysis methods was beneficial for evaluating outbreak measures developed through emergency response planning initiatives that consider the managed movement of hatching eggs from monitored premises in an HPAI Control Area.

Incorporating risk communication into highly pathogenic avian influenza preparedness and response efforts.

A highly pathogenic avian influenza (HPAI) outbreak in the United States will initiate a federal emergency response effort that will consist of disease control and eradication efforts, including quarantine and movement control measures. These movement control measures will not only apply to live animals but also to animal products. However, with current egg industry "just-in-time" production practices, limited storage is available to hold eggs. As a result, stop movement orders can have significant unintended negative consequences, including severe disruptions to the food supply chain. Because stakeholders' perceptions of risk vary, waiting to initiate communication efforts until an HPAI event occurs can hinder disease control efforts, including the willingness of producers to comply with the response, and also can affect consumers' demand for the product. A public-private-academic partnership was formed to assess actual risks involved in the movement of egg industry products during an HPAI event through product specific, proactive risk assessments. The risk analysis process engaged a broad representation of stakeholders and promoted effective risk management and communication strategies before an HPAI outbreak event. This multidisciplinary team used the risk assessments in the development of the United States Department of Agriculture, Highly Pathogenic Avian Influenza Secure Egg Supply Plan, a comprehensive response plan that strives to maintain continuity of business. The collaborative approach that was used demonstrates how a proactive risk communication strategy that involves many different stakeholders can be valuable in the development of a foreign animal disease response plan and build working relationships, trust, and understanding.

The impact of holding time on the likelihood of moving internally contaminated eggs from a highly pathogenic avian influenza infected but undetected commercial table-egg layer flock.

Emergency response during a highly pathogenic avian influenza (HPAI) outbreak may involve quarantine and movement controls for poultry products such as eggs. However, such disease control measures may disrupt business continuity and impact food security, since egg production facilities often do not have sufficient capacity to store eggs for prolonged periods. We propose the incorporation of a holding time before egg movement in conjunction with targeted active surveillance as a novel approach to move eggs from flocks within a control area with a low likelihood of them being contaminated with HPAI virus. Holding time reduces the likelihood of HPAI-contaminated eggs being moved from a farm before HPAI infection is detected in the flock. We used a stochastic disease transmission model to estimate the HPAI disease prevalence, disease mortality, and fraction of internally contaminated eggs at various time points postinfection of a commercial table-egg layer flock. The transmission model results were then used in a simulation model of a targeted matrix gene real-time reverse transcriptase (RRT)-PCR testing based surveillance protocol to estimate the time to detection and the number of contaminated eggs moved under different holding times. Our simulation results indicate a significant reduction in the number of internally contaminated eggs moved from an HPAI-infected undetected flock with each additional day of holding time. Incorporation of a holding time and the use of targeted surveillance have been adopted by the U.S. Department of Agriculture in their Draft Secure Egg Supply Plan for movement of egg industry products during an HPAI outbreak.

Impact of virus strain characteristics on early detection of highly pathogenic avian influenza infection in commercial table-egg layer flocks and implications for outbreak control.

Early detection of highly pathogenic avian influenza (HPAI) infection in commercial poultry flocks is a critical component of outbreak control. Reducing the time to detect HPAI infection can reduce the risk of disease transmission to other flocks. The timeliness of different types of detection triggers could be dependent on clinical signs that are first observed in a flock, signs that might vary due to HPAI virus strain characteristics. We developed a stochastic disease transmission model to evaluate how transmission characteristics of various HPAI strains might effect the relative importance of increased mortality, drop in egg production, or daily real-time reverse transcriptase (RRT)-PCR testing, toward detecting HPAI infection in a commercial table-egg layer flock. On average, daily RRT-PCR testing resulted in the shortest time to detection (from 3.5 to 6.1 days) depending on the HPAI virus strain and was less variable over a range of transmission parameters compared with other triggers evaluated. Our results indicate that a trigger to detect a drop in egg production would be useful for HPAI virus strains with long infectious periods (6-8 days) and including an egg-drop detection trigger in emergency response plans would lead to earlier and consistent reporting in some cases. We discuss implications for outbreak control and risk of HPAI spread attributed to different HPAI strain characteristics where an increase in mortality or a drop in egg production or both would be among the first clinical signs observed in an infected flock.

Biosecurity on a multiple-age egg production complex: a 15-year experience.

A multiple-age egg production farm was conceived, designed, constructed, and managed with the goal of blocking the introduction of preventable poultry diseases and infestations. Fifteen years after conception and 13 years after housing the first hens, the 4 million-hen farm remains free of Mycoplasma gallisepticum, infectious laryngotracheitis, and Ornithonyssus sylviarum. Annual savings in vaccine use exceed the estimated annual costs of the biosecurity program. The cumulative design, construction, and operation of this farm are a successful demonstration of practical, effective biosecurity on a commercial poultry farm.

Prevalence of low pathogenicity avian influenza virus during 2005 in two U.S. live bird market systems.

Oropharyngeal and cloacal swabs were collected from poultry sold in two live bird market (LBM) systems to estimate the prevalence of low pathogenicity avian influenza virus (LPAIV) shedding during the summer and fall of 2005. Random sampling was conducted in three LBMs in Minnesota where 50 birds were sampled twice weekly for 4 wk, and in three LBMs in a California marketing system. A stratified systematic sampling method was used to collect samples from Southern California LBMs, where LPAIV was detected during routine surveillance. No LPAIV was detected in the LBM system in Minnesota where realtime reverse transcription-PCR (RT-PCR) was conducted on oropharyngeal samples. RT-PCR was performed on swabs taken from 290 of 14,000, 65 of 252, and 60 of 211 birds at the three Southern California LBMs. The number of samples collected was based on the number of birds, age of the birds, and number of species present in the LBM. Virus isolation, subtyping, and sequencing of the hemagglutinin, neuraminidase, and other internal protein genes was performed on AIV-positive samples. The estimated prevalence of LPAIV in California was 0.345% in an LBM/supply farm with multiple ages of Japanese quail, 3% in an LBM with multiple ages and strains of chickens present, and 49.8% in an LBM with multiple species, multiple strains, and multiple ages. The positive virus samples were all LPAIV H6N2 and closely related to viruses isolated from Southern California in 2001 and 2004. Little or no comingling of poultry may contribute to little or no LPAIV detection in the LBMs.

A Retrospective Study of Early vs. Late Virus Detection and Depopulation on Egg Laying Chicken Farms Infected with Highly Pathogenic Avian Influenza Virus During the 2015 H5N2 Outbreak in the United States.

The 2015 highly pathogenic avian influenza (HPAI) H5N2 outbreak affected more than 200 Midwestern U.S. poultry premises. Although each affected poultry operation incurred substantial losses, some operations of the same production type and of similar scale had differences between one another in their ability to recognize evidence of the disease before formal diagnoses and in their ability to make proactive, farm-level disease containment decisions. In this case comparison study, we examine the effect of HPAI infection on two large egg production facilities and the epidemiologic and financial implications resulting from differences in detection and decision-making processes. Each egg laying facility had more than 1 million caged birds distributed among 18 barns on one premises (Farm A) and 17 barns on the other premises (Farm B). We examine how farm workers' awareness of disease signs, as well as how management's immediate or delayed decisions to engage in depopulation procedures, affected flock mortality, levels of environmental contamination, time intervals for re population, and farm profits on each farm. By predictive mathematical modeling, we estimated the time of virus introduction to examine how quickly infection was identified on the farms and then estimated associated contact rates within barns. We found that the farm that implemented depopulation immediately after detection of abnormal mortality (Farm A) was able to begin repopulation of barns 37 days sooner than the farm that began depopulation well after the detection of abnormally elevated mortality (Farm B). From average industry economic data, we determined that the loss associated with delayed detection using lost profit per day in relation to down time was an additional $3.3 million for Farm B when compared with Farm A.

Estudio retrospectivo de detección viral temprana y tardía y despoblación en granjas de gallinas de postura infectadas con el virus de la influenza aviar altamente patógeno durante el brote de H5N2 del año 2015 en los Estados Unidos. El brote de influenza aviar altamente patógena (HPAI) H5N2 del año 2015 afectó a más de 200 granjas avícolas del medio oeste de los Estados Unidos. Aunque cada operación avícola afectada incurrió en pérdidas sustanciales, algunas operaciones del mismo tipo de producción y de escala similar tuvieron diferencias entre sí en su capacidad para reconocer evidencias de la enfermedad antes de los diagnósticos formales y en su capacidad para realizar decisiones proactivas para la contención de la enfermedad a nivel de granja. En este estudio de caso, se examinó el efecto de la infección por influenza aviar altamente patógena en dos instalaciones grandes de producción de huevo y las implicaciones epidemiológicas y financieras que fueron resultado de los diferentes procesos de detección y toma de decisiones. Cada instalación de postura de huevo tenía más de un millón de aves enjauladas distribuidas en 18 casetas en una granja (Granja A) y 17 casetas en las otras instalaciones (Granja B). Se examinó cómo el conocimiento de los trabajadores agrícolas sobre los signos de la enfermedad, así como cómo las decisiones de manejo inmediatas o tardías para establecer procedimientos de despoblación, afectaron la mortalidad de las parvadas, los niveles de contaminación ambiental, los intervalos de tiempo para la repoblación y las ganancias en cada granja. Mediante un modelo matemático predictivo, se estimó el tiempo de introducción del virus para examinar la rapidez con la que se identificó la infección en las granjas y luego se estimaron las tasas de contacto asociadas dentro de las casetas. Se encontró que la granja que implementó la despoblación inmediatamente después de la detección de mortalidad anormal (Granja A) pudo comenzar la repoblación de las casetas 37 días antes que la granja que comenzó la despoblación mucho después de la detección de mortalidad anormalmente elevada (Granja B). A partir de los datos económicos promedio de la industria, se determinó que la pérdida asociada con la detección tardía utilizando las pérdidas de ganancias por día en relación con el tiempo de inactividad fue de $3.3 millones adicionales para la Granja B en comparación con la Granja A.

Simulated Flock-Level Shedding Characteristics of Turkeys in Ten Thousand Bird Houses Infected with H7 Low Pathogenicity Avian Influenza Virus Strains.

Understanding the amount of virus shed at the flock level by birds infected with low pathogenicity avian influenza virus (LPAIV) over time can help inform the type and timing of activities performed in response to a confirmed LPAIV-positive premises. To this end, we developed a mathematical model which allows us to estimate viral shedding by 10,000 turkey toms raised in commercial turkey production in the United States, and infected by H7 LPAIV strains. We simulated the amount of virus shed orally and from the cloaca over time, as well as the amount of virus in manure. In addition, we simulated the threshold cycle value (Ct) of pooled oropharyngeal swabs from birds in the infected flock tested by real-time reverse transcription polymerase chain reaction. The simulation model predicted that little to no shedding would occur once the highest threshold of seroconversion was reached. Substantial amounts of virus in manure (median 1.5×108 and 5.8×109; 50% egg infectious dose) were predicted at the peak. Lastly, the model results suggested that higher Ct values, indicating less viral shedding, are more likely to be observed later in the infection process as the flock approaches recovery.

Surveillance and Sequestration Strategies to Reduce the Likelihood of Transporting Highly Pathogenic Avian Influenza Virus Contaminated Layer Manure.

Movement and land application of manure is a known risk factor for secondary spread of avian influenza viruses. During an outbreak of highly pathogenic avian influenza (HPAI), movement of untreated (i.e., fresh) manure from premises known to be infected is prohibited. However, moving manure from apparently healthy (i.e., clinically normal) flocks may be critical, because some egg-layer facilities have limited on-site storage capacity. The objective of this analysis was to evaluate targeted dead-bird active surveillance real-time reverse transcriptase polymerase chain reaction (rRT-PCR) testing protocols that could be used for the managed movement of manure from apparently healthy egg-layer flocks located in an HPAI control area. We also evaluated sequestration, which is the removal of manure from any contact with chickens, or with manure from other flocks, for a period of time, while the flock of origin is actively monitored for the presence of HPAI virus. We used stochastic simulation models to predict the chances of moving a load of contaminated manure, and the quantity of HPAI virus in an 8 metric ton (8000 kg) load of manure moved, before HPAI infection could be detected in the flock. We show that the likelihood of moving contaminated manure decreases as the length of the sequestration period increases from 3 to 10 days (e.g., for a typical contact rate, with a sample pool size of 11 swabs, the likelihood decreased from 48% to <1%). The total quantity of feces from HPAI-infectious birds in a manure load moved also decreases. Results also indicate that active surveillance protocols using 11 swabs per pool result in a lower likelihood of moving contaminated manure relative to protocols using five swabs per pool. Simulation model results from this study are useful to inform further risk evaluation of HPAI spread through pathways associated with manure movement and further evaluation of biosecurity measures intended to reduce those risks.

Artículo regular­Estrategias de vigilancia y aislamiento para reducir la probabilidad de transportar gallinaza de aves de postura contaminada con virus de influenza aviar altamente patógeno. El movimiento y la aplicación de gallinaza a la tierra es un factor de riesgo conocido para la propagación secundaria de los virus de la influenza aviar (IA). Durante un brote de influenza aviar altamente patógena (IAAP), se prohíbe el movimiento de gallinaza sin tratar (es decir, fresco) de las instalaciones que se conoce que están infectadas. Sin embargo, el traslado de gallinaza de parvadas aparentemente sanas (es decir, clínicamente normales) puede ser fundamental, porque algunas instalaciones de producción de huevo tienen una capacidad limitada de almacenamiento en el lugar. El objetivo de este análisis estaba evaluar los protocolos de la prueba de transcriptasa reversa y reacción en cadena de la polimerasa en tiempo real (rRT-PCR) utilizados en la vigilancia activa dirigida a aves muertas, que podrían usarse para el movimiento controlado de gallinaza de parvadas de postura aparentemente sanas ubicadas en un área de control para influenza aviar de alta patogenicidad. También se evaluó el aislamiento, que es la remoción de gallinaza y prevenir cualquier contacto con pollos, o con gallinaza de otras parvadas, durante un período de tiempo, mientras que la parvada de origen es monitoreada activamente para detectar la presencia del virus de la influenza aviar altamente patógeno. Se utilizaron modelos de simulación estocástica para predecir las posibilidades de trasladar una carga de estiércol contaminado y la cantidad de virus de la influenza aviar altamente patógeno en una carga de ocho toneladas métricas (8000 kg) de gallinaza trasladada, antes de que se pudiera detectar la infección por influenza aviar altamente patógena en la parvada. Se demostró que la probabilidad de mover gallinaza contaminada disminuye a medida que la duración del período de aislamiento aumenta de tres a diez días (por ejemplo, para una tasa de contacto típica, con un tamaño de muestra de 11 hisopos, la probabilidad disminuyó de 48% a <1 %). La cantidad total de heces de aves infectadas por la influenza aviar altamente patógena en una carga de gallinaza transportada también disminuye. Los resultados también indican que los protocolos de vigilancia activa que utilizan 11 hisopos como muestra agrupada dan como resultado una menor probabilidad de mover gallinaza contaminada en comparación con los protocolos que utilizan cinco hisopos por muestra agrupada. Los resultados del modelo de simulación de este estudio son útiles para una evaluación adicional del riesgo de la propagación de la influenza aviar altamente patógena a través de vías asociadas con el movimiento de gallinaza y una evaluación adicional de las medidas de bioseguridad destinadas a reducir esos riesgos.

Stability of the glycoprotein gene of avian metapneumovirus (Canada goose isolate 15a/01) after serial passages in cell cultures.

The glycoprotein (G) gene sequences of avian metapneumovirus (aMPV) subtypes A, B, C, and D are variable in size and number of nucleotides. The G gene of early U.S. turkey isolates of aMPV-C have been reported to be 1798 nucleotides (nt) (585 aa) in length, whereas the G genes of more recent turkey isolates have been reported to be 783 nucleotides. In some studies, the G gene of aMPV-C turkey isolates was found to be truncated to a smaller G gene of 783 nt (261 aa) upon serial passages in Vero cells. This is believed to be due to the deletion of 1015 nt near the end of the open reading frame. The purpose of this study was to determine variation, if any, in the G gene of an aMPV-C isolated from a wild bird (Canada goose [Branta canadensis]) following serial passages in Vero cells. No size variation was observed for up to 50 passages, except for a few amino acid changes in the extracellular domain at the 50th passage level. The G gene of this wild bird isolate appears to be unique from subtype C metapneumoviruses of turkeys.

Role of Clostridium perfringens and Clostridium septicum in causing turkey cellulitis.

The role of Clostridium perfringens and Clostridium septicum in the development of cellulitis and mortality in turkey poults was examined. Studies were done in turkeys of two age groups: 3-wk-old and 7-wk-old turkey poults. The effect of varying doses of C. perfringens and C. septicum in reproducing cellulitis lesions and mortality in turkeys was investigated. Both in vitro and in vivo assays were conducted to study their toxic and biologic activities. Clostridium septicum spore culture was found to be more potent than that of C. perfringens in both in vitro assays, such as the hemolysis test, and in vivo assays in mice and turkeys. Both C. perfringens and C. septicum spore cultures were found to be capable of inducing cellulitis lesions and mortality in turkey poults when inoculated by subcutaneous route. Histopathology examination of affected tissues revealed a "moth-eaten appearance, with abundant growth of C. perfringens and C. septicum in the sarcomeres of muscle tissues and in the subcutaneous tissues. However, C. septicum was found to be more potent than C. perfringens in causing cellulitis lesions and mortality in turkeys. Three-week-old poults were found to be less susceptible than 7-wk-old poults in the development of cellulitis lesions and mortality after inoculation with either spore cultures of C. perfringens or C. septicum. The results of the current study suggest that although C. septicum is more potent in causing cellulitis lesions and mortality, infection with either C. septicum or C. perfringens can cause cellulitis lesions and mortality in turkeys.

Conducting Influenza Virus Pathogenesis Studies in Avian Species.

Avian infection studies with influenza A are an important means of assessing host susceptibility, viral pathogenesis, host responses to infection, mechanisms of transmission, viral pathotype, and viral evolution. Complex systems and natural settings may also be explored with carefully designed infection studies. In this chapter, we explore the elements of infection studies, general guidelines for choosing a virus to use, host selection, and many aspects of study design.

Environmental Sampling for Influenza A Viruses in Turkey Barns.

We have examined a variety of sampling strategies for detecting pathogens in turkey flocks undergoing infections with low pathogenicity avian influenza virus (LPAIV). We found that viral RNA was widely distributed in the barn environment of turkey flocks undergoing an active LPAIV infection and was in both water and drinker biofilm samples. Viral RNA was concentrated in drinker biofilm and sediment and was detectable using real-time reverse-transcription polymerase chain reaction (RRT-PCR) and by virus isolation. Drinker biofilm sample results correlated with concurrently collected oropharyngeal (OP) sample results from flocks on a farm with LPAI in which the two sampling strategies were directly compared. To evaluate the utility of biofilm sampling for the detection of highly pathogenic avian influenza virus (HPAIV), biofilm and OP swabs from mortality pools were collected daily from negative turkey flocks on an HPAI-positive premise. The biofilm swabs were positive 1-2 days prior to positives appearing in the OP sample pools. The drinker biofilm sampling strategy overcame the difficulty of finding a subclinical infectious bird in a population by collecting material from a large number of individuals and testing a sample in which a positive signal persists for several days to weeks. The sampling method is convenient for use in turkey barns and has been reliably used in both active and passive surveillance programs for LPAIV and HPAIV using RRT-PCR.

Muestreo ambiental para el virus de influenza A en casetas de pavos. Se han examinado una variedad de estrategias de muestreo para detectar patógenos en parvadas de pavos que sufren infecciones con el virus de la influenza aviar de baja patogenicidad (con las siglas en inglés LPAIV). Se encontró que el ARN viral se distribuyó ampliamente en el ambiente de las casetas con parvadas de pavos con infección activa por el virus de la influenza aviar de baja patogenicidad y se determinó tanto en muestras de agua como en muestras de la biopelícula de bebederos. El ARN viral se concentró en la biopelícula y en el sedimento de bebederos y se detectó mediante transcripción reversa y reacción en cadena de la polimerasa en tiempo real (RRT-PCR) y mediante el aislamiento del virus. Los resultados de la muestra de la biopelícula del bebedero se correlacionaron con los resultados de la muestra orofaríngea (OP) colectada de forma simultánea de parvadas en una granja con influenza aviar de baja patogenicidad en las que se compararon directamente las dos estrategias de muestreo. Para evaluar la utilidad del muestreo de la biopelícula para la detección del virus de la influenza aviar altamente patógena (HPAIV), se recolectaron diariamente biopelículas e hisopos orofaríngeos de grupos de mortalidad de parvadas de pavos negativas en una granja positiva para la influenza aviar de alta patogenicidad. Los hisopos de biopelículas fueron positivos de uno a dos días antes de que aparecieran resultados positivos en las muestras orofaríngeas agrupadas. La estrategia de muestreo de la biopelícula del bebedero eliminó la dificultad de encontrar un ave infectada subclínicamente en una población al recolectar material de un gran número de individuos y analizar una muestra en la que persiste una señal positiva durante varios días o semanas. El método de muestreo es adecuado para su uso en casetas de pavos y se ha utilizado de manera confiable en los programas de vigilancia activa y pasiva para el virus de influenza aviar tanto de baja como de alta patogenicidad utilizando transcripción reversa y reacción en cadena de la polimerasa en tiempo real.

Mortality-Based Triggers and Premovement Testing Protocols for Detection of Highly Pathogenic Avian Influenza Virus Infection in Commercial Upland Game Birds.

Outbreaks involving avian influenza viruses are often devastating to the poultry industry economically and otherwise. Disease surveillance is critically important because it facilitates timely detection and generates confidence that infected birds are not moved during business continuity intended to mitigate associated economic losses. The possibility of using an abnormal increase in daily mortality to levels that exceed predetermined thresholds as a trigger to initiate further diagnostic investigations for highly pathogenic avian influenza (HPAI) virus infection in the flock is explored. The range of optimal mortality thresholds varies by bird species, trigger type, and mortality thresholds, and these should be considered when assessing sector-specific triggers. The study uses purposefully collected data and data from the literature to determine optimal mortality triggers for HPAI detection in commercial upland game bird flocks. Three trigger types were assessed for the ability to detect rapidly both HPAI (on the basis of disease-induced and normal mortality data) and false alarm rate (on the basis of normal mortality data); namely, 1) exceeding a set absolute threshold on one day, 2) exceeding a set absolute threshold on two consecutive days, or 3) exceeding a multiple of a seven-day moving average. The likelihood of disease detection using some of these triggers together with premovement real-time reverse transcription PCR (rRT-PCR) testing was examined. Results indicate that the performance of the two consecutive days trigger had the best metrics (i.e., rapid detection with few false alarms) in the trade-off analysis. The collected normal mortality data was zero on 66% of all days recorded, with an overall mean of 0.6 dead birds per day. In the surveillance scenario analyses, combining the default protocol that relied only on active surveillance (i.e., premovement testing of oropharyngeal swab samples from dead birds by rRT-PCR) together with either of the mortality-based triggers improved detection rates on all days postexposure before scheduled movement. For exposures occurring within 8 days of movement, the protocol that combined the default with single-day triggers had slightly more detections than that with two consecutive days triggers. However, all assessed protocol combinations were able to detect all infections that occurred more than 10 days before scheduled movement. These findings can inform risk-based decisions pertaining to continuity of business in the commercial upland game bird industry.

Activadores basados en la mortalidad y protocolos de pruebas de premovimiento para la detección de la infección del virus de influenza aviar altamente patógena en aves de caza de tierras altas comerciales Los brotes que involucran virus de influenza aviar a menudo son económicamente devastadores para la industria avícola. La vigilancia de enfermedades es de importancia crítica porque facilita la detección oportuna y genera confianza en que las aves infectadas no serán movilizadas para continuar con la operación de las industrias avícolas para mitigar las pérdidas económicas asociadas. Se explora la posibilidad de utilizar un aumento anormal en la mortalidad diaria a niveles que excedan umbrales predeterminados como un desencadenante para iniciar investigaciones de diagnóstico para la infección del virus de la influenza aviar altamente patógena en la parvada. El rango de umbrales de mortalidad óptimos varían según la especie de ave, el tipo de activador y los umbrales de mortalidad y estos deben considerarse al evaluar los activadores específicos del sector. El estudio utiliza datos recopilados de manera planeada y datos de la literatura para determinar los desencadenantes de mortalidad óptimos para la detección de la influenza aviar altamente patógena en las parvadas comerciales de aves de caza de tierras altas. Se evaluaron tres activadores de acuerdo a su capacidad de detectar rápidamente influenza aviar altamente patógena (en función de los datos de mortalidad normal e inducida por la enfermedad) y la tasa de falsa alarma (en función de los datos de mortalidad normal); como son, 1) que se exceda un umbral absoluto establecido en un día, 2) que se exceda un umbral absoluto establecido en dos días consecutivos, o 3) que excede un múltiplo de un promedio móvil de siete días. Se examinó la probabilidad de detección de la enfermedad utilizando algunos de estos desencadenantes junto con la prueba de transcripción reversa y PCR en tiempo real (rRT-PCR). Los resultados indicaron que el rendimiento del disparador de dos días consecutivos tuvo los mejores resultados (es decir, detección rápida con pocas falsas alarmas) en el análisis costo-beneficio. Los datos de mortalidad normal recopilados fueron cero en el 66% de todos los días registrados, con una media general de 0.6 aves muertas por día. En los análisis de escenarios de vigilancia, la combinación del protocolo predeterminado que se basó únicamente en la vigilancia activa (por ejemplo pruebas antes de movilizaciones con muestras de hisopos orofaríngeos por rRT-PCR de aves muertas) a la par con cualquiera de los desencadenantes basados en la mortalidad mejoraron las tasas de detección en todos los días posteriores a la exposición antes del movimiento programado. Para las exposiciones que ocurrieron dentro de los ocho días de movimiento, el protocolo que combinó el valor predeterminado con los activadores de un solo día tuvo un poco más de detecciones que el de los activadores de dos días consecutivos. Sin embargo, todas las combinaciones de protocolos evaluadas pudieron detectar todas las infecciones que ocurrieron por más de 10 días antes del movimiento programado. Estos hallazgos pueden proveer información para la toma de decisiones basadas en el riesgo relacionadas con la continuidad de operaciones en la industria comercial de aves de caza de tierras altas.

Evaluating the Effect of the Within-Flock Disease Transmission Rate on Premovement Active Surveillance in Low Pathogenicity Avian Influenza-Infected Flocks.

Premovement active surveillance for low pathogenicity avian influenza (LPAI) may be a useful risk management tool for producers during high-risk periods, such as during an LPAI outbreak, or in areas where there is a recognized high risk for LPAI spread. The effectiveness of three active-surveillance protocols in mitigating LPAI spread risk related to the movement of spent broiler breeders to processing was evaluated in this study. Each protocol differed in the amount of real-time reverse transcription polymerase chain reaction (RRT-PCR) and serology testing conducted. The protocols were evaluated with the use of disease transmission and active surveillance simulation models parametrized specifically for broiler breeders to estimate the probability of detecting a current or past infection and the mean proportion of infectious birds at the time of sampling in houses where the infection remains undetected at the time of movement after exposure to the virus. The two values were estimated considering flock infection for 1-28 days prior to the day of scheduled movement. A distribution for the adequate contact rate, a parameter that controls the rate of within-house spread in the disease transmission model, was estimated for this study by a novel forward simulation approach with the use of serology data from three LPAI-infected broiler breeder flocks in the United States. The estimated distribution suggests that the lower contact-rate estimates from previously published studies were not a good fit for the serology results observed in these U.S. flocks, though considerable uncertainty remains in the parameter estimate. The results for the probability of detection and mean proportion of infectious, undetected birds suggest that RRT-PCR testing is most beneficial during the early stages of infection postexposure, and serology testing is most beneficial during the later stages of infection, results that are expected to hold for flocks outside the United States as well. Thus, protocols that combine RRT-PCR and serology testing can offer a more balanced approach with good performance over the disease course in a flock.

Evaluación del efecto de la tasa de transmisión dentro de la parvada en la vigilancia activa previa al movimiento de parvadas infectadas por influenza aviar de baja patogenicidad. La vigilancia activa para la influenza aviar de baja patogenicidad (LPAI) previa al movimiento puede ser una herramienta útil en el manejo de riesgos para los productores durante períodos de alto riesgo, como durante un brote de influenza aviar de baja patogenicidad o en áreas donde se reconoce que existe un alto riesgo de propagación de esta enfermedad. En este estudio, se evaluó la efectividad de tres protocolos de vigilancia activa para mitigar el riesgo de propagación de la influenza aviar de baja patogenicidad relacionado con el movimiento de los reproductores pesados de desecho a la planta de procesamiento. Los protocolos diferían en la cantidad de muestras procesadas por la transcriptasa reversa y reacción en cadena de la polimerasa en tiempo real (rRT-PCR) y por las pruebas serológicas realizadas. Los protocolos se evaluaron utilizando modelos de simulación de vigilancia activa y transmisión de la enfermedad con parámetros específicamente para reproductores pesados, para estimar la probabilidad de detectar una infección actual o pasada y la proporción media de aves con infección activa al momento del muestreo en casetas donde la infección permanecía sin detectar al momento del movimiento después de la exposición al virus. Los dos valores se estimaron considerando la infección de la parvada de uno a 28 días antes de la fecha programada para el movimiento. Una distribución para la tasa de contacto adecuada, un parámetro que controla la tasa de propagación dentro de la caseta en el modelo de transmisión de la enfermedad, se estimó para este estudio mediante un novedoso enfoque de simulación directa utilizando datos serológicos de tres parvadas reproductores pesados infectados con influenza aviar de baja patogenicidad en los Estados Unidos. La distribución estimada sugiere que las estimaciones de la tasa de contacto más baja obtenida de los estudios publicados previamente no fueron una buena opción para los resultados serológicos observados en estas parvadas en los Estados Unidos, aunque sigue existiendo una gran incertidumbre en la estimación del parámetro. Los resultados de la probabilidad de detección y la proporción media de aves con infección no detectadas sugieren que la prueba rRT-PCR es más beneficiosa durante las primeras etapas de la infección después de la exposición, mientras que la serología es más beneficiosa durante las últimas etapas de la infección, resultados que se espera apliquen también para parvadas fuera de los Estados Unidos. Por lo tanto, los protocolos que combinan rRT-PCR y las pruebas de serología pueden ofrecer un enfoque más equilibrado con un buen rendimiento durante el curso de la enfermedad en una parvada.

Comparative pathogenicity of early and recent isolates of avian metapneumovirus subtype C in turkeys.

The objective of the present study was to compare the pathogenicity of early and recent isolates of avian metapneumovirus subtype-C (aMPV-C) in turkeys. Two-week-old turkeys were inoculated with early and recent isolates of aMPV-C. Clinical signs were monitored. Tissues were examined for viral ribonucleic acid (RNA), lesions, and viral antigen by reverse transcription-polymerase chain reaction (RT-PCR), histopathology and immunohistochemistry, respectively. Birds infected with the recent isolate had higher clinical sign scores than those infected with the early isolate. Only the recent isolate produced a multifocal loss of cilia in the nasal turbinate of infected birds. Immunohistochemistry revealed intense staining of aMPV antigen in turbinate and trachea of birds infected with the recent isolate. The findings indicate that the recent isolate produced more severe clinical signs and lesions in turkeys compared to the early isolate. The recent isolate could be ideal for the development of a challenge model for aMPV infection in turkeys.

Spatial transmission of H5N2 highly pathogenic avian influenza between Minnesota poultry premises during the 2015 outbreak.

The spatial spread of highly pathogenic avian influenza (HPAI) H5N2 during the 2015 outbreak in the U.S. state of Minnesota was analyzed through the estimation of a spatial transmission kernel, which quantifies the infection hazard an infectious premises poses to an uninfected premises some given distance away. Parameters were estimated using a maximum likelihood method for the entire outbreak as well as for two phases defined by the daily number of newly detected HPAI-positive premises. The results indicate both a strong dependence of the likelihood of transmission on distance and a significant distance-independent component of outbreak spread for the overall outbreak. The results further suggest that HPAI spread differed during the later phase of the outbreak. The estimated spatial transmission kernel was used to compare the Minnesota outbreak with previous HPAI outbreaks in the Netherlands and Italy to contextualize the Minnesota transmission kernel results and make additional inferences about HPAI transmission during the Minnesota outbreak. Lastly, the spatial transmission kernel was used to identify high risk areas for HPAI spread in Minnesota. Risk maps were also used to evaluate the potential impact of an early marketing strategy implemented by poultry producers in a county in Minnesota during the outbreak, with results providing evidence that the strategy was successful in reducing the potential for HPAI spread.