Evaluation of Feedstuffs as a Potential Carrier of Avian Influenza Virus between Feed Mills and Poultry Farms.

The present study was conducted to assess the potential vector role of feedstuffs for the area spreading of avian influenza virus (AIV). Firstly, feed samples were collected from commercial poultry facilities that experienced highly pathogenic avian influenza (H5N2) in 2014−2015 for AIV testing by a real-time RT−PCR specific for the viral matrix gene. Secondly, feed materials obtained from an AIV-negative farm were spiked with various concentrations of a low pathogenic AIV H5N2. Virus-spiked cell culture media were prepared in the same manner and used for comparison. The spiked feed and media samples were tested by a multiplex real-time RT−PCR ran in a quantitative manner, either immediately or after incubation at −20, 4, 22, and 37 °C for 24, 48, and 72 h. Some of the feedstuffs collected from the poultry facilities or feed mills were positive for AIV RNA but negative by the virus isolation (VI) test, while all the formaldehyde-treated feedstuffs were PCR-negative. In the spiked feeds, the AIV titer was 1−3 logs lower than that in the corresponding media, even when tested immediately after spiking, suggesting that feed might have a negative impact on the virus or PCR detection. The half-life of AIV RNA was shorter at a higher temperature. A significant decay in the viral RNA over time was noted at 37 °C (p < 0.05), suggesting that feedstuffs should be maintained in the cold chain when testing is desired. Furthermore, the thermal degradation of AIV suggests that the heat treatment of feeds could be an alternative to chemical treatment when contamination is suspected. Collectively, the study observations indicate that AIV survivability in feed is relatively low, thus rendering it a low risk.

Structure and immune recognition of the porcine epidemic diarrhea virus spike protein.

Porcine epidemic diarrhea virus (PEDV) is an alphacoronavirus responsible for significant morbidity and mortality in pigs. A key determinant of viral tropism and entry, the PEDV spike protein is a key target for the host antibody response and a good candidate for a protein-based vaccine immunogen. We used electron microscopy to evaluate the PEDV spike structure, as well as pig polyclonal antibody responses to viral infection. The structure of the PEDV spike reveals a configuration similar to that of HuCoV-NL63. Several PEDV protein-protein interfaces are mediated by non-protein components, including a glycan at Asn264 and two bound palmitoleic acid molecules. The polyclonal antibody response to PEDV infection shows a dominance of epitopes in the S1 region. This structural and immune characterization provides insights into coronavirus spike stability determinants and explores the immune landscape of viral spike proteins.

Environmental Sampling for Avian Influenza Virus Detection in Commercial Layer Facilities.

The present study was designed to evaluate the utility of environmental samples for convenient but accurate detection of avian influenza virus (AIV) in commercial poultry houses. First, environmental samples from AIV-negative commercial layer facilities were spiked with an H5N2 low pathogenic AIV and were evaluated for their effect on the detection of viral RNA immediately or after incubation at -20 C, 4 C, 22 C, or 37 C for 24, 48, or 72 hr. Second, Swiffer pads, drag swabs, and boot cover swabs were evaluated for their efficiency in collecting feces and water spiked with the H5N2 LPAIV under a condition simulated for a poultry facility floor. Third, environmental samples collected from commercial layer facilities that experienced an H5N2 highly pathogenic AIV outbreak in 2014-15 were evaluated for the effect of sampling locations on AIV detection. The half-life of AIV was comparable across all environmental samples but decreased with increasing temperatures. Additionally, sampling devices did not differ significantly in their ability to collect AIV-spiked environmental samples from a concrete floor for viral RNA detection. Some locations within a poultry house, such as cages, egg belts, house floor, manure belts, and manure pits, were better choices for sampling than other locations (feed trough, ventilation fan, and water trays) to detect AIV RNA after cleaning and disinfection. Samples representing cages, floor, and manure belts yielded significantly more PCR positives than the other environmental samples. In conclusion, environmental samples can be routinely collected from a poultry barn as noninvasive samples for monitoring AIV.

Muestreo ambiental para la detección del virus de la influenza aviar en instalaciones de aves de postura comerciales. El presente estudio fue diseñado para evaluar la utilidad de las muestras ambientales para la detección rápida pero precisa del virus de la influenza aviar (AIV) en casetas avícolas comerciales. Primero, muestras ambientales de las instalaciones comerciales de aves de postura negativas para influenza aviar se inocularon con un virus de la influenza de baja patogenicidad (LPAIV) H5N2 y se evaluaron para determinar su efecto en la detección de ARN viral inmediatamente o después de la incubación a -20 C, 4 C, 22 C o 37 C durante 24 hr, 48 hr o 72 horas. En segundo lugar, se evaluaron las esponjas marca Swiffer, los hisopos de arrastre y los cubre botas para muestreo ambiental para determinar su eficiencia en la recolección de heces y agua inoculada con el virus de influenza aviar de baja patogenicidad H5N2 en una condición simulada de piso de una instalación avícola. En tercer lugar, muestras ambientales recolectadas de instalaciones comerciales de ponedoras que experimentaron un brote de influenza aviar altamente patógena H5N2 en 2014-15, se evaluaron para determinar el efecto de la ubicación de muestreo en la detección de influenza aviar. La vida media del virus de la influenza aviar fue comparable en todas las muestras ambientales, pero disminuyó con el aumento de la temperatura. Además, los dispositivos de muestreo no difirieron significativamente en su capacidad para recolectar muestras ambientales inoculadas con influenza aviar de un piso de concreto para la detección de ARN viral. Algunas ubicaciones dentro de la caseta aviar, como jaulas, bandas transportadoras de huevo, piso de la caseta, bandas transportadoras de gallinasa y fosas de gallinasa, fueron las mejores opciones para el muestreo en comparación con otras ubicaciones (comederos, ventiladores y bandejas de agua) para detectar el ARN del virus de influenza después de la limpieza y desinfección. Las muestras que representan jaulas, piso y bandas transportadoras de gallinasa arrojaron significativamente más resultados positivos de PCR que las otras muestras ambientales. En conclusión, las muestras ambientales se pueden recolectar rutinariamente de uns granja avícola como muestras no invasivas para monitorear al virus de influenza aviar.

Induction of Reactive Intermediates and Autophagy-Related Proteins upon Infection of Macrophages with Rhodococcus equi.

Rhodococcus equi (R. equi) is an intracellular macrophage-tropic pathogen with potential for causing fatal pyogranulomatous pneumonia in foals between 1 and 6 months of age. In this study, we sought to determine whether infection of macrophages with R. equi could lead to the induction of autophagy. Murine bone marrow derived macrophages (BMDM) were infected with R. equi for various time intervals and analyzed for upregulation of autophagy proteins and accumulation of autophagosomes relative to uninfected controls. Western blot analysis showed a progressive increase in LC3-II and Beclin1 levels in a time-dependent manner. The functional accumulation of autophagosomes detected with monodansylcadaverine further supported the enhanced induction of autophagy in BMDM infected with R. equi. In addition, infection of BMDM with R. equi induced generation of reactive oxygen species (ROS) in a time-dependent manner. These data are consistent with reports documenting the role of ROS in induction of autophagy and indicate that the infection of macrophages by R. equi elicits innate host defense mechanisms.