Duration of Highly Pathogenic Avian Influenza Virus and Newcastle Disease Virus Infectivity in Dried Ornithologic Study Skins.

Ornithologic study skins are specimens of avian skins that have been preserved by drying after removing the viscera and muscle. Because of the high value of study skins for scientific studies, specimens are shared among researchers. There is concern that study skins might be contaminated with high-consequence diseases such as highly pathogenic avian influenza virus (HPAIV) or Newcastle disease virus (NDV). To mitigate risk, thermal or chemical treatment of study skins may be required before transfer; however, such treatments might damage the specimens. Therefore, a study was conducted to evaluate the duration of infectivity of HPAIV and NDV in study skins prepared from infected chickens (Gallus gallus). Study skins were prepared from 10 chickens infected with each virus. Skin and feather pulp samples were taken at the time of study skin preparation to establish starting titers. Mean starting titers in skins were 4.2 log10 and 5.1 log10 50% egg infectious doses (EID50) for HPAIV and NDV groups respectively, and were 6.7 log10 EID50 for HPAIV, and 6.4 log10 EID50 for NDV in feather pulp. Samples were collected at 2 and 4 wk of drying to quantify viable virus. At 2 wk, fewer samples had detectable virus and mean titers were 1.8 log10 (skin) and 2.1 log10 (feathers) EID50 for HPAIV, and 1.7 log10 (skin) and 3.5 log10 (feathers) EID50 for NDV. At 4 wk viable virus could not be detected in either tissue type.

The Thermal Stability of Newcastle Disease Virus in Poultry Litter.

Sanitary disposal of contaminated organic material during recovery from an animal disease outbreak is costly and laborious. Characterizing the thermal stability of avian paramyxovirus type 1 (APMV-1; virulent APMV-1 strains cause Newcastle disease in poultry) will help inform risk assessments on the presence of viable virus on infected premises or in organic waste from infected premises. In some environments and housing types, heat may also be used as a decontamination method. Therefore, the objective of this study was to characterize the thermal stability (i.e., decimal reduction values [D values]) of APMV-1 in poultry litter. Virus inactivation was evaluated at seven temperatures from 10.0 C through 43.3 C, at 5.5 C intervals (50-110 F in 10 F intervals), using the I2 isolate of APMV-1, a vaccine strain known to be thermally stable. A high titer of virus (approximately 108 50% egg infectious doses) was added to wood shavings based, soiled chicken litter (poultry litter). Litter with both low and high moisture levels were evaluated. Samples were collected at different time intervals, and infectious virus was titrated in embryonated chicken eggs. At high temperatures (37.8 C-43.3 C), infectious virus could not be detected after 2-7 days, whereas at lower temperatures (10 C-21.1 C), it took up to 112 days for virus to decrease to undetectable levels. Furthermore, the D values were almost always shorter in the high moisture litter.

Estabilidad térmica del virus de la enfermedad de Newcastle en la cama avícola. La eliminación sanitaria de material orgánico contaminado durante la recuperación de un brote de enfermedad en los animales es costosa y laboriosa. La caracterización de la estabilidad térmica del paramixovirus aviar tipo 1 (APMV-1; las cepas virulentas de APMV-1 que causan la enfermedad de Newcastle en avicultura) contribuirá con información para las evaluaciones de riesgo sobre la presencia de virus viables en las instalaciones infectadas o en los desechos orgánicos de las instalaciones infectadas. En algunos entornos y tipos de casetas, el calor también se puede utilizar como método de descontaminación. Por lo tanto, el objetivo de este estudio fue caracterizar la estabilidad térmica (es decir, valores de reducción decimal [valores D]) del APMV-1 en la cama de aves. La inactivación del virus se evaluó a siete temperaturas, desde 10.0 C hasta 43.3 C, en intervalos de 5.5 C (50­110 F en intervalos de 10 F), utilizando el aislamiento I2 de APMV-1, que es una cepa vacunal conocida por ser térmicamente estable. Se añadió el virus con un título alto (aproximadamente 108 dosis infecciosas de embrión de pollo al 50%) a la cama de pollo con base de virutas de madera (cama avícola). Se evaluaron camas con niveles de humedad altos y bajos. Se recolectaron muestras en diferentes intervalos de tiempo y se tituló el virus infeccioso en huevos embrionados de pollo. A altas temperaturas (37.8 C­43.3 C), el virus infeccioso no se pudo detectar después de dos a siete días, mientras que a temperaturas más bajas (10 C­21.1 C), el virus tardó hasta 112 días en disminuir a niveles no detectables. Además, los valores D fueron casi siempre más cortos en la cama con alta humedad.

Optimizing sample collection methods for detection of respiratory viruses in poultry housing environments.

Viral respiratory diseases, such as avian influenza, Newcastle disease, infectious bronchitis and infectious laryngotracheitis, have considerable negative economic implications for poultry. Ensuring the virus-free status of premises by environmental sampling after cleaning and disinfection is essential for lifting a quarantine and/or safely restocking the premises following an outbreak. The objectives of this study were to identify optimal sample collection devices and to determine the locations in poultry housing which are best for poultry respiratory virus sample collection. Chickens exposed to infectious bronchitis virus, which was used as a representative virus for enveloped poultry respiratory viruses, were housed in floor-pens in either a curtain-sided wood framed house or a cement block house. Foam swabs, cellulose sponges, polyester swabs, dry cotton gauze and pre-moistened cotton gauze were evaluated for comparative efficiency in recovering viral RNA. Cotton gauze pre-moistened with the viral transport media had the highest sensitivity among the devices (wood-framed house: 78% positive, geometric mean titre [GMT] of 2.6 log10 50% egg infectious doses [EID50 ] equivalents/ml; cement block houses: 55% positive, GMT of 1.7 log10 EID50 equivalents/ml). Targeting virus deposition sites is also crucial for efficient virus elimination procedures and subsequent testing; therefore, 10 locations within the houses were compared for virus detection. In both housing types, the highest viral RNA loads were recovered from the tops of drinker lines within the pen. Places the chickens could contact directly (e.g., feeder rim) or were contacted by caretaker feet (hallway floor) also yielded higher levels of viral RNA more consistently. These results will facilitate the establishment of efficient environmental sampling procedures for respiratory viruses of poultry.

Identification of optimal sample collection devices and sampling locations for the detection of environmental viral contamination in wire poultry cages.

Environmental testing of poultry premises after an outbreak of an infectious disease like avian influenza (AI) or Newcastle disease is essential to promptly verify virus-free status and subsequently return to normal operations. In an attempt to establish an optimized sampling protocol, a laboratory study simulating an AI virus-contaminated poultry house with wire layer cages was conducted. Three sample collection devices, pre-moistened cotton gauze, dry cotton gauze and a foam swab, were evaluated with each of four sample locations within a cage and when sampling all four locations with one device. Virus was detected with quantitative real-time RT-PCR utilizing a standard curve of a quantified homologous isolate of AI virus to determine titre equivalents of virus. The pre-moistened gauze detected the most virus RNA (100% positive, geometric mean titre [GMT): 3.2 log10 50% embryo infectious doses [EID50 ] equivalents per 25 cm2 ) in all four sample locations compared to dry gauze (93% positive, GMT: 2.6 EID50 equivalents per 25 cm2 ) and foam swabs (95% positive, GMT: 2.8 log10 EID50 equivalents per 25 cm2 ). The highest viral RNA loads were observed from the cage floor, and when the four locations were sampled with the same device. Overall, the pre-moistened gauze performed the best, and sampling multiple locations within a cage with the same device would likely optimize detection of residual virus.

Identification of Efficacious Vaccines Against Contemporary North American H7 Avian Influenza Viruses.

Five vaccines, including four inactivated, whole-virus water-in-oil adjuvanted vaccines and a commercial nonreplicating alphavirus-vectored RNA particle (RP) vaccine were evaluated in chickens for their ability to provide protection against challenge with a recent H7 highly pathogenic avian influenza virus (AIV) from the United States (A/turkey/IN/1403-1/2016 H7N8). One of the inactivated vaccines and the RP vaccine were prepared with A/turkey/IN/16-01571-6/2016 H7N8 low pathogenic AIV (LPAIV; TK/IN/16), which is identical to the challenge virus, except for the proteolytic cleavage site of the hemagglutinin protein. The remaining three inactivated vaccines were prepared with other North American H7 LPAIVs. The hemagglutination inhibition assay was used to evaluate the antigenic relationships among the vaccines and selected recent H7 AIV isolates. All five vaccines provided protection against mortality. The inactivated vaccines reduced virus shedding significantly at 2 and 4 days post challenge compared with sham-vaccinated chickens. In contrast, the RP vaccine did not significantly reduce virus shedding. The inactivated vaccine prepared with TK/IN/16 elicited the highest antibody responses, which suggests it is a strong candidate for use as an antigen for North American H7 AIVs. Antigenic distance calculations showed that the four inactivated vaccine strains and other recent North American H7 isolates are antigenically similar, which suggests that the vaccines evaluated here would be similar enough to provide protection to other North American H7 AIVs. If future H7 outbreaks in poultry warrant vaccination, the field strain can be rapidly evaluated with these antigens and, if adequately related, one of these characterized strains may be used.

Artículo reguarl­Identificación de vacunas eficaces contra los virus contemporáneos de la influenza aviar H7 de América del Norte. Se evaluaron cinco vacunas, incluidas cuatro vacunas inactivadas con virus completo y con adyuvante de agua en aceite y una vacuna comercial de partículas de ARN en un vector de alfavirus no replicante (RP) en pollos para determinar su capacidad para brindar protección contra el desafío con un virus de influenza aviar altamente patógena H7 (AIV) de los Estados Unidos (A/pavo/IN/1403-1/2016 H7N8). Una de las vacunas inactivadas y la vacuna de partículas de ARN se prepararon con un virus de influenza aviar de baja patogenicidad A/pavo/IN/16-01571-6/2016 AIV de baja patogenicidad H7N8 (LPAIV; TK/IN/16), que es idéntico al virus de desafío, excepto por el sitio de disociación proteolítica de la proteína hemaglutinina. Las tres vacunas inactivadas restantes se prepararon con otros virus de baja patogenicidad H7 de América del Norte. El ensayo de inhibición de la hemaglutinación se utilizó para evaluar las relaciones antigénicas entre las vacunas y los aislados del virus de influenza aviar H7 recientes seleccionados. Las cinco vacunas proporcionaron protección contra la mortalidad. Las vacunas inactivadas redujeron significativamente la diseminación del virus a los 2 y 4 días posteriores al desafío en comparación con los pollos que recibieron la vacunación falsa. Por el contrario, la vacuna de partículas de ARN no redujo significativamente la diseminación del virus. La vacuna inactivada preparada con el virus TK/IN/16 provocó las respuestas de anticuerpos más altas, lo que indica que es un fuerte candidato para su uso como antígeno contra los virus de influenza aviar H7 de América del Norte. Los cálculos de la distancia antigénica mostraron que las cuatro cepas de vacunas inactivadas y otros aislados recientes del subtipo H7 de América del Norte son antigénicamente similares, lo que sugiere que las vacunas evaluadas en este estudio serían lo suficientemente similares para brindar protección a otros virus de influenza aviar de H7 de América del Norte. Si futuros brotes de H7 en la avicultura justifican la vacunación, la cepa de campo se puede evaluar rápidamente con estos antígenos y si están adecuadamente relacionados, se puede utilizar una de estas cepas caracterizadas.

Effects of an H7 Highly Pathogenic and Related Low Pathogenic Avian Influenza Virus on Chicken Egg Production, Viability, and Virus Contamination of Egg Contents and Surfaces.

Both highly pathogenic (HP) and low pathogenic (LP) avian influenza virus (AIV) can cause decreases or even cessation of egg production in chickens and turkeys. Production of abnormal eggs (deformed, thin-shelled, soft-shelled) can also be caused by AIV infection. Additionally, egg surfaces and contents may also be contaminated with virus. Because data quantifying these effects are lacking, white Plymouth Rock hens were inoculated with HP or LP AIV while in production. No decreases in egg production or abnormal eggs were observed with LPAIV-infected hens. No lesions or viral antigen staining in ovary and oviduct were observed in LPAIV-infected hens 3 days postchallenge. LPAIV RNA was detected on eggs collected from 12 hr to 11 days postinoculation (PI) and was on or in 6.4% (15/234) of the eggs. Titer equivalents of LPAIV ranged from 1.3-2.5 log10 50% egg infectious doses (EID50). No virus was detected in embryo tissue from eggs laid by LPAIV-infected hens. In contrast, egg production by HPAIV-inoculated hens decreased at 72 hr PI and 18.4% (16/87) of the eggs were abnormal. However, viability was similar to that of the sham inoculates. HPAIV RNA was detected in or on 11.1% (9/81) of the eggs from 36 hr through 96 hr PI, when the hens were euthanatized. HPAIV RNA was detected on 6.2% of eggshells, in 4.2% of albumin/yolk samples, and in 8.3% of embryo tissue. Forty percent of the abnormal eggs were positive for HPAIV RNA. Titer equivalents on or in HPAIV-contaminated eggs ranges from 1.0-4.0 log10 EID50. Lesions and viral antigen staining were present in the ovary and all sections of the oviduct of infected hens 3 days postchallenge. These data will inform models using production-based triggers for LPAIV monitoring and for risk assessments to determine the disposition of eggs from flocks infected with LPAIV or HPAIV.

Efectos de un virus de la influenza aviar altamente patógeno y un virus de baja patogenicidad H7 relacionados en la producción de huevos de gallina, la viabilidad y con la contaminación viral del contenido y las superficies del huevo. Tanto el virus de la influenza aviar (AIV) altamente patógeno (HP) como el de baja patogenicidad (LP) pueden causar una disminución o incluso el cese de la producción de huevos en pollos y pavos. La producción de huevos anormales (con cascaron deforme o delgado, o con cascarón blando) también puede ser causada por la infección por el virus de la influenza aviar. Además, las superficies y el contenido del huevo también pueden estar contaminados con virus. Debido a la falta de datos que cuantifiquen estos efectos, gallinas Plymouth Rock blancas fueron inoculadas con virus de alta o baja patogenicidad mientras estaban en producción. No se observaron disminuciones en la producción de huevos o huevos anormales en las gallinas infectadas con el virus de influenza aviar de baja patogenicidad. No se observaron lesiones ni la presencia del antígeno viral en el ovario y oviducto de gallinas infectadas con el virus de baja patogenicidad a los tres días después del desafío. El ARN del virus de baja patogenicidad se detectó en los huevos recolectados de 12 horas a 11 días después de la inoculación y estaba sobre o dentro del 6.4% (15/234) de los huevos. Los títulos equivalentes del virus de baja patogenicidad variaron de 1.3 a 2.5 log10 dosis infecciosas 50% para embrión de huevo (EID50). No se detectó virus en el tejido embrionario de los huevos puestos por gallinas infectadas con el virus de alta patogenicidad. En contraste, la producción de huevos por gallinas inoculadas con el virus de alta disminuyó a las 72 horas después de la inoculación y el 18.4% (16/87) de los huevos fueron anormales. Sin embargo, la viabilidad fue similar a la de los controles negativos inoculados. Se detectó ARN del virus de alta patogenicidad en el 11.1% (9/ 81) de los huevos desde las 36 horas hasta 96 horas después de la inoculación, cuando las gallinas se sacrificaron. Se detectó ARN del virus de alta patogenicidad en el 6.2% de los cascarones de huevo, en el 4.2% de las muestras de albúmina/yema y en el 8.3% del tejido embrionario. Cuarenta por ciento de los huevos anormales fueron positivos para ARN del virus de alta patogenicidad. Los títulos equivalentes sobre o por dentro de los huevos contaminados con influenza aviar de alta patogenicidad variaron de 1.0­4.0 log10 EID50. Las lesiones y la presencia del antígeno viral estaban presentes en el ovario y en todas las secciones del oviducto de las gallinas infectadas tres días después del desafío. Estos datos servirán como fuente de información para los modelos que utilizan disparadores basados en la producción para el monitoreo del virus de influenza aviar de baja patogenicidad y para las evaluaciones de riesgos para determinar la eliminación de los huevos de las parvadas infectadas con virus de influenza aviar de baja o de alta patogenicidad.

Assessment of replicate numbers for titrating avian influenza virus using dose-response models.

Embryonating chicken eggs (ECEs) are among the most sensitive laboratory host systems for avian influenza virus (AIV) titration, but ECEs are expensive and require space for storage and incubation. Therefore, reducing ECE use would conserve resources. We utilized statistical modeling to evaluate the accuracy and precision of AIV titration with 3 instead of 5 ECEs for each dilution by the Reed-Muench method for 50% endpoint calculation. Beta-Poisson and exponential dose-response models were used in a simulation study to evaluate observations from actual titration data from 18 AIV isolates. The reproducibility among replicates of a titration was evaluated with one AIV isolate titrated in 3 replicates with the beta-Poisson, exponential, and Weibull dose-response models. The standard deviation (SD) of the error between input and estimated virus titers was estimated with Monte Carlo simulations using the fitted dose-response models. Good fit was observed with all models that were utilized. Reducing the number of ECEs per dilution from 5 to 3 resulted in the width of the 95% confidence interval increasing from ±0.64 to ±0.75 log10 50% ECE infectious doses (EID50) and the SD of the error increased by 0.03 log10 EID50. Our study suggests that using fewer ECEs per dilution is a viable approach that will allow laboratories to reduce costs and improve efficiency.

Clade 2.3.4.4 H5 North American Highly Pathogenic Avian Influenza Viruses Infect, but Do Not Cause Clinical Signs in, American Black Ducks (Anas rubripes).

Highly pathogenic avian influenza virus (HPAIV) from the goose/Guangdong/1996 clade 2.3.4.4 H5 lineage spread from Asia into North America in 2014, most likely by wild bird migrations. Although several variants of the virus were detected, H5N8 and H5N2 were the most widespread in North American wild birds and domestic poultry. In early 2015, the H5N2 virus spread through commercial poultry in the Midwest, and >50 million chickens and turkeys died or had to be culled. Related H5 HPAIVs are still endemic in much of the Eastern Hemisphere. The wild bird species that were involved with dissemination of the virus in North America are not known. Dabbling ducks, especially mallards (Anas platyrhynchos), typically have the highest detection rates for avian influenza viruses. To better characterize the wild avian species that could spread the virus, American black ducks (Anas rubripes), which are closely related to mallards, were challenged with the North American H5N2 and H5N8 index HPAIV isolates: A/Northern Pintail/WA/40964/2014 H5N2 and A/Gyrfalcon/WA/41088/2014 H5N8. Although the American black ducks could be infected with low doses of both isolates (≤102 50% egg infective doses), ducks shed the H5N2 longer than the H5N8 (10 vs. 7 days) and the titers of virus shed were higher. Although there were too few ducks available on which to draw definitive conclusions, this suggests that American black ducks could serve as a more efficient reservoir for the H5N2 virus than the H5N8 virus.

Nota de investigación- Los virus de influenza aviar altamente patógenos de América del Norte H5 clado 2.3.4.4 son capaces de infectar pero no causan signos clínicos en ánades sombríos americanos (Anas rubripes). Los virus de la influenza aviar altamente patógena (HPAIV) subtipo H5pertenecientes al linaje ganso/Guangdong/1996 clado 2.3.4.4, se han propagado desde Asia a América del Norte en el año 2014, muy probablemente por migración de aves silvestres. Aunque se detectaron varias variantes del virus, los subtipos H5N8 y H5N2 fueron los más extendidas en aves silvestres y aves domésticas de América del Norte. A principios de 2015, el virus H5N2 se propagó a través de aves comerciales en el medio oeste de los Estados Unidos, y más de 50 millones de pollos y pavos murieron o tuvieron que ser eliminados. Virus de la influenza aviar de alta patogenicidad H5 relacionados aún son endémicos en gran parte del hemisferio oriental. Las especies de aves silvestres que participaron en la diseminación del virus en América del Norte no se conocen. Los patos chapoteadores, especialmente los patos silvestres de collar, suelen tener las tasas de detección más altas para los virus de la influenza aviar. Para caracterizar mejor las especies de aves silvestres que podrían propagar el virus, ánades sombríos americanos (Anas rubripes), que están estrechamente relacionados con los patos silvestres, se desafiaron con aislamientos índices del virus de la influenza aviar de alta patogenicidad de América del Norte H5N2 y H5N8: A/ánade rabudo/WA/40964/2014 H5N2 o con el virus A/halcón gerifalte/WA/41088/2014 H5N8. Aunque los ánades sombríos americanos podieron infectarse con dosis bajas de ambos aislamientos (≤102 50% de dosis infectivas para embrión de pollo), los patos eliminaron al virus H5N2 por más tiempo en comparación con el virus H5N8 (10 días y 7 días, respectivamente) y los títulos de virus fueron más altos. Aunque había muy pocos patos disponibles para sacar conclusiones definitivas, esto sugiere que los ánades sombríos americanos podrían servir como un reservorio más eficiente para el virus H5N2.

The Pathogenesis of H7 Highly Pathogenic Avian Influenza Viruses in Lesser Scaup (Aythya affinis).

Waterfowl are the natural hosts of avian influenza virus (AIV), and through migration spread the virus worldwide. Most AIVs carried by wild waterfowl are low pathogenic strains; however, Goose/Guangdong/1996 lineage clade 2.3.4.4 H5 highly pathogenic (HP) AIV now appears to be endemic in wild birds in much of the Eastern Hemisphere. Most research efforts studying AIV pathogenicity in waterfowl thus far have been directed toward dabbling ducks. In order to better understand the role of diving ducks in AIV ecology, we previously characterized the pathogenesis of clade 2.3.4.4 H5 HPAIV in lesser scaup (Aythya affinis). In an effort to further elucidate AIV infection in diving ducks, the relative susceptibility and pathogenesis of two North American lineage H7 HPAIV isolates from the most recent outbreaks in the United States was investigated. Lesser scaup were inoculated with either A/turkey/IN/1403-1/2016 H7N8 or A/chicken/TN/17-007147-2/2017 H7N9 HPAIV by the intranasal route. The approximate 50% bird infectious dose (BID50) of the H7N8 isolate was determined to be 103 50% egg infectious doses (EID50), and the BID50 of the H7N9 isolate was determined to be <102 EID50, indicating some variation in adaptation between the two isolates. No mortality or clinical disease was observed in either group except for elevated body temperatures at 2 and 4 days postinoculation (DPI). Virus shedding was detected up to 14 DPI from both groups, and there was a trend for shedding to have a longer duration and at higher titer levels from the cloacal route. These results demonstrate that lesser scaup are susceptible to both H7 lineages of HPAIV, and similar to dabbling duck species, they shed virus for long periods relative to gallinaceous birds and don't present with clinical disease.

Nota de investigación- Patogenia de los virus de influenza aviar altamente patógenos H7 en porrones bola (Aythya affinis). Las aves acuáticas son los hospedadores naturales del virus de la influenza aviar y a través de la migración, propagan el virus en todo el mundo. La mayoría de los virus de influenza transportados por aves acuáticas silvestres son cepas de baja patogenicidad; sin embargo, actualmente el virus altamente patogénico Ganso/Guangdong/1996 linaje 2.3.4.4 H5 parece ser endémico en aves silvestres en gran parte del hemisferio oriental. La mayor parte de los esfuerzos de investigación que estudian la patogenicidad del virus de influenza aviar en aves acuáticas hasta ahora se han dirigido a los patos chapuceadores. Para comprender mejor el papel de los patos buceadores en la ecología del virus de influenza aviar, se caracterizó previamente la patogenia del virus de alta patogenicidad clado 2.3.4.4 H5 en porrones bola (Aythya affinis). En un esfuerzo por dilucidar aún más la infección por el virus de influenza aviar en patos buceadores, se investigó la susceptibilidad relativa y la patogénesis de dos aislamientos H7 de alta patogenicidad con linaje de América del Norte que circularon en brotes recientes en los Estados Unidos. Los porrones bola se inocularon con los virus de alta patogenicidad A/pavo/IN/1403-1/2016 H7N8 o con el virus A/pollo/N/17-007147-2/2017 H7N9 por vía intranasal. Se determinó que la dosis infecciosa aproximada de aves 50% (BID50) del aislado H7N8 era de 103 dosis infecciosas embrión de pollo 50% (EID50), y se determinó que el BID50 del aislado H7N9 era <102 EID50, lo que indica alguna variación en la adaptación entre dos aislamientos. No se observó mortalidad ni enfermedad clínica en ninguno de los grupos, con excepción de elevadas temperaturas corporales a los dos y cuatro días posteriores a la inoculación. La eliminación del virus se detectó hasta 14 días después de la inoculación en ambos grupos y hubo una tendencia de que la eliminación por la ruta cloacal mostrara una mayor duración y niveles más altos de títulos. Estos resultados demuestran que el porrón bola es susceptible a ambos linajes H7 del virus de la influenza aviar de alta patogenicidad y similar a las especies de patos chapuceadores, eliminan el virus durante largos períodos en relación con las aves gallináceas y no presentan enfermedad clínica.

Efficacy of novel recombinant fowlpox vaccine against recent Mexican H7N3 highly pathogenic avian influenza virus.

Since 2012, H7N3 highly pathogenic avian influenza (HPAI) has produced negative economic and animal welfare impacts on poultry in central Mexico. In the present study, chickens were vaccinated with two different recombinant fowlpox virus vaccines (rFPV-H7/3002 with 2015 H7 hemagglutinin [HA] gene insert, and rFPV-H7/2155 with 2002 H7 HA gene insert), and were then challenged three weeks later with H7N3 HPAI virus (A/chicken/Jalisco/CPA-37905/2015). The rFPV-H7/3002 vaccine conferred 100% protection against mortality and morbidity, and significantly reduced virus shed titers from the respiratory and gastrointestinal tracts. In contrast, 100% of sham and rFPV-H7/2155 vaccinated birds shed virus at higher titers and died within 4 days. Pre- (15/20) and post- (20/20) challenge serum of birds vaccinated with rFPV-H7/3002 had antibodies detectable by hemagglutination inhibition (HI) assay using challenge virus antigen. However, only a few birds (3/20) in the rFPV-H7/2155 vaccinated group had antibodies that reacted against the challenge strain but all birds had antibodies that reacted against the homologous vaccine antigen (A/turkey/Virginia/SEP-66/2002) (20/20). One possible explanation for differences in vaccines efficacy is the antigenic drift between circulating viruses and vaccines. Molecular analysis demonstrated that the Mexican H7N3 strains have continued to rapidly evolve since 2012. In addition, we identified in silico three potential new N-glycosylation sites on the globular head of the H7 HA of A/chicken/Jalisco/CPA-37905/2015 challenge virus, which were absent in 2012 H7N3 outbreak virus. Our results suggested that mutations in the HA antigenic sites including increased glycosylation sites, accumulated in the new circulating Mexican H7 HPAIV strains, altered the recognition of neutralizing antibodies from the older vaccine strain rFPV-H7/2155. Therefore, the protective efficacy of novel rFPV-H7/3002 against recent outbreak Mexican H7N3 HPAIV confirms the importance of frequent updating of vaccines seed strains for long-term effective control of H7 HPAI virus.

The Effect of Infectious Bursal Disease Virus-Induced Immunosuppression on Vaccination Against Highly Pathogenic Avian Influenza Virus.

Poor efficacy of avian influenza virus (AIV) vaccines in chickens has been documented in the field in spite of good results in experimental settings. Although the causes are multifactorial and complex, one contributing factor may be prior infection with immunosuppressive viruses. In an effort to evaluate the role of immunosuppressive agents on AIV pathogenesis and vaccine efficacy, the effect of prior infection with infectious bursal disease virus (IBDV), a ubiquitous immunosuppressive virus of chickens, was evaluated. Specific-pathogen-free white Plymouth Rock chickens were exposed to variant E IBDV at 1 day of age and were subsequently vaccinated with an inactivated H7 AIV vaccine 2 wk later. Vaccinated chickens exposed to IBDV had a geometric mean antibody titer to AIV of 1:1.7 by hemagglutination inhibition assay compared to a geometric mean titer of 1:47.5 from chickens that were vaccinated but not exposed to IBDV. Three weeks postvaccination, the chickens were challenged with one of six different doses of highly pathogenic (HP) AIV homologous to the vaccine. Within challenge virus dose groups, vaccinated chickens exposed to IBDV had similar mortality rates to nonvaccinated chickens that were not exposed to IBDV. In contrast, vaccinated chickens that were not exposed to IBDV were protected from mortality. Exposure to IBDV also decreased the mean death time (2.3-3.7 days depending on dose) compared with vaccinated birds not exposed to IBDV (4-7 days depending on dose). Neither vaccination nor IBDV infection had an effect on mean bird infection dose with HPAIV, but the 50% bird lethal dose was reduced from >106 50% egg infective dose (EID50) in the vaccinated, IBDV-nonexposed group to 103.3 EID50 in the vaccinated group exposed to IBDV. These results are consistent with IBDV exposure contributing to poor vaccine efficacy in the field.

Thermal Inactivation of avian influenza virus in poultry litter as a method to decontaminate poultry houses.

Removal of contaminated material from a poultry house during recovery from an avian influenza virus (AIV) outbreak is costly and labor intensive. Because AIV is not environmentally stable, heating poultry houses may provide an alternative disinfection method. The objective was to determine the time necessary to inactivate AIV in poultry litter at temperatures achievable in a poultry house. Low pathogenic (LP) AIV inactivation was evaluated between 10.0°-48.9°C, at ∼5.5°C intervals and highly pathogenic (HP) AIV inactivation was evaluated between 10.0°-43.3°C, at ∼11°C intervals. Samples were collected at numerous time points for each temperature. Virus isolation in embryonating chicken eggs was conducted to determine if viable virus was present. Each sample was also tested by real-time RT-PCR. Low pathogenicity AIV was inactivated at 1day at 26.7°C or above. At 10.0, 15.6 and 21.1°C, inactivation times increased to 2-5days. Highly pathogenic AIV followed a similar trend; the virus was inactivated after 1day at 43.3°C and 32.2°C, and required 2 and 5days for inactivation at 21.1°C and 10.0°C respectively. While low pathogenicity AIV appeared to be inactivated at a lower temperature than high pathogenicity AIV, this was not due to any difference in the strains, but due to fewer temperature points being evaluated for high pathogenicity. Endpoints for detection by real-time RT-PCR were not found even weeks after the virus was inactivated. This provides a guideline for the time required, at specific temperatures to inactivate AIV in poultry litter and likely on surfaces within the house. Heat treatment will provide an added level of safety to personnel and against further spread by eliminating infectious virus prior to cleaning a house.

The pathogenesis of H7N8 low and highly pathogenic avian influenza viruses from the United States 2016 outbreak in chickens, turkeys and mallards.

In January 2016, a combined outbreak of highly pathogenic (HP) avian influenza virus (AIV) and low pathogenicity (LP) AIV occurred in commercial turkeys in the state of Indiana, United States. Genetically, the viruses were highly similar, belonged to the North American wild bird lineage, and had not been previously detected in poultry. In order to understand the pathobiology of the H7N8 LPAIV and HPAIV, infectivity, transmission and pathogenicity studies were conducted in chickens, turkeys, and mallards. Among the three species the lowest mean infectious dose for both the LP and HP phenotype was for turkeys, and also disease from the LPAIV was only observed with turkeys. Furthermore, although the HPAIV was lethal for both chickens and turkeys, clinical signs caused by the HPAIV isolate differed between the two species; neurological signs were only observed in turkeys. Mallards could be infected with and transmit both viruses to contacts, but neither caused clinical disease. Interestingly, with all three species, the mean infectious dose of the HP isolate was at least ten times lower than that of the LP isolate. This study corroborates the high susceptibility of turkeys to AIV as well as a pathobiology that is different from chickens. Further, this study demonstrates that mallards can be asymptomatically infected with HP and LP AIV from gallinaceous poultry and may not just be involved in transmitting AIV to them.

THE PATHOGENESIS OF CLADE 2.3.4.4 H5 HIGHLY PATHOGENIC AVIAN INFLUENZA VIRUSES IN RUDDY DUCK (OXYURA JAMAICENSIS) AND LESSER SCAUP (AYTHYA AFFINIS).

Waterfowl are the natural hosts of avian influenza virus (AIV) and disseminate the virus worldwide through migration. Historically, surveillance and research efforts for AIV in waterfowl have focused on dabbling ducks. The role of diving ducks in AIV ecology has not been well characterized. In this study, we examined the relative susceptibility and pathogenicity of clade 2.3.4.4 H5 highly pathogenic AIV (HPAIV) in two species of diving ducks. Juvenile and adult Ruddy Duck (Oxyura jamaicensis) and juvenile Lesser Scaup (Aythya affinis) were intranasally inoculated with A/Northern Pintail/WA/40964/2014 H5N2 HPAIV. Additional groups of juvenile Lesser Scaups were inoculated with A/Gyrfalcon/WA/41088/2014 H5N8 HPAIV. The approximate 50% bird infectious doses (BID50) of the H5N2 isolate for adult Ruddy Ducks was <102 50% egg infectious doses (EID50) and for the juvenile Lesser Scaups it was <104 EID50. There were insufficient juvenile Ruddy Ducks to calculate the BID50. The BID50 for the juvenile Lesser Scaups inoculated with the H5N8 isolate was 103 EID50. Clinical disease was not observed in any group; however, mortality occurred in the juvenile Ruddy Ducks inoculated with the H5N2 virus (three of five ducks), and staining for AIV antigen was observed in numerous tissues from these ducks. One adult Ruddy Duck also died and although it was infected with AIV (the duck was positive for virus shedding and AIV antigen was detected in tissues), it was also infected with coccidiosis. The proportion of ducks shedding virus was related to the dose administered, but the titers were similar among dose groups. The group with the fewest ducks shedding virus was the adult Ruddy Ducks. There was a trend for the Lesser Scaups to shed higher titers of virus than the Ruddy Ducks. No virus shedding was detected after 7 d postinoculation in any group. Similar to dabbling ducks, Lesser Scaups and Ruddy Ducks are susceptible to infection with this H5 HPAIV lineage, although they excrete lower titers of virus.