Brian J. Jordan

Avian-Induced Pluripotent Stem Cells Derived Using Human Reprogramming Factors

Avian species are important model animals for developmental biology and disease research. However, unlike in mice, where clonal lines of pluripotent stem cells have enabled researchers to study mammalian gene function, clonal and highly proliferative pluripotent avian cell lines have been an elusive goal. Here we demonstrate the generation of avian induced pluripotent stem cells (iPSCs), the first nonmammalian iPSCs, which were clonally isolated and propagated, important attributes not attained in embryo-sourced avian cells. This was accomplished using human pluripotency genes rather than avian genes, indicating that the process in which mammalian and nonmammalian cells are reprogrammed is a conserved process. Quail iPSCs (qiPSCs) were capable of forming all 3 germ layers in vitro and were directly differentiated in culture into astrocytes, oligodendrocytes, and neurons. Ultimately, qiPSCs were capable of generating live chimeric birds and incorporated into tissues from all 3 germ layers, extraembryonic tissues, and potentially the germline. These chimera competent qiPSCs and in vitro differentiated cells offer insight into the conserved nature of reprogramming and genetic tools that were only previously available in mammals.

Vaccination against infectious bronchitis virus: A continuous challenge

Infectious bronchitis virus (IBV) is a significant respiratory pathogen of commercial poultry that causes millions of dollars in lost revenue worldwide each year. Even though the poultry industry extensively vaccinates against IBV, emergence of new serotypes and variants continually occur, making control of the disease difficult. Current mass application strategies for IBV vaccines are inefficient and frequently result in vaccination failures. Novel vaccine technology development has been slow, and is hindered by the constraints of large-scale poultry production. Further complicating the situation is the lack of knowledge of IBV protein and host cell interactions, making targeted vaccine intervention strategies near impossible. Taken together, it is easy to see why this disease remains significant in poultry production. This review outlines the current situation as it relates to IBV control, including vaccination, vaccines, and development of immunity, and recent developments in vaccine technology that may provide better protection in the future.

Detection of Infectious Bronchitis Virus with the Use of Real-Time Quantitative Reverse Transcriptase-PCR and Correlation with Virus Detection in Embryonated Eggs

SUMMARY Real-time quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) assays have been used to detect the presence of challenge virus when the efficacy of infectious bronchitis virus (IBV) vaccine against field viruses is being experimentally evaluated. However, federal guidelines for licensing IBV vaccines indicate that challenge-virus detection following vaccination is to be conducted in embryonated eggs. In this study, we examined qRT-PCR data with the use of universal and type-specific primers and probe sets for IBV detection and compared those data with challenge-virus detection in embryonated eggs to determine if the two methods of evaluating vaccine efficacy are comparable. In addition, we tested the qRT-PCR assays on thermocyclers from two different manufacturers. We found the universal IBV primers and probe set to be comparable to challenge-virus detection in embryonated eggs. However, for some IBV types (Mass41 and Conn on the SmartCycler II and Ark, Mass41, Conn, and GA98 on the ABI 7500) the qRT-PCR assay was more sensitive than virus detection in embryonated eggs. This may simply be due to the universal IBV qRT-PCR assay being more sensitive than virus detection in eggs or to the assay detecting nucleic acid from nonviable virus. This finding is important and needs to be considered when evaluating challenge-virus detection for vaccination and challenge studies, because qRT-PCR could potentially identify positive birds that would otherwise be negative by virus detection in embryonated eggs; thus it could lead to a more stringent measure of vaccine efficacy. We also found that the IBV type-specific primers and probe sets designed in this study were in general less sensitive than the universal IBV primers and probe set. Only the Ark-DPI–specific assay on the SmartCycler II and the Ark-DPI–, Mass41–, and DE072/GA98– (for detection of GA98 virus only) specific assays on the ABI 7500 were comparable in sensitivity to virus detection in eggs. We found that a number of variables, including the virus type examined, primers and probe efficiency and stability, and assay conditions, including thermocycler platform, can affect the data obtained from qRT-PCR assays. These results indicate that qRT-PCR assays can be used to detect IBV challenge virus, but each assay, including the assay conditions and thermocycler, should be individually evaluated if those data are expected to be comparable to virus detection in embryonated eggs. RESUMEN Detección del virus de la bronquitis infecciosa mediante métodos de transcripción reversa y PCR en tiempo real cuantitativos y su correlación con la detección del virus en huevos embrionados. Los ensayos de transcripción reversa y reacción en cadena de la polimerasa en tiempo real (qRT-PCR) se han utilizado para detectar la presencia de virus de desafío cuando se evalúa experimentalmente la eficacia de virus vacunales de la bronquitis infecciosa (IBV) contra virus de campo. Sin embargo, los lineamientos federales para la concesión de licencias de vacunas contra la bronquitis infecciosa indican que la detección del virus de desafío después de la vacunación se debe llevar a cabo en huevos embrionados. En este estudio se analizaron los datos por qRT-PCR con el uso de conjuntos de iniciadores y de sondas universales y específicos para la detección del virus de bronquitis y se compararon estos datos con la detección del virus en huevos embrionados para determinar si ambos métodos son comparables en la evaluación de la eficacia de las vacunas. Además, se analizaron los ensayos de qRT-PCR en termocicladores de dos fabricantes diferentes. Se encontró que los iniciadores y sondas universales para bronquitis infecciosa son comparables a la detección de virus en huevos embrionados. Sin embargo, para algunos tipos del virus de bronquitis (Mass41 y Conn con SmartCycler II y Ark, Mass41, Conn, y GA98 con el ABI 7500) el ensayo de qRT-PCR fue más sensible que la detección del virus en huevos embrionados. Esto puede ser debido simplemente a que el ensayo de qRT-PCR universal es más sensible que la detección en huevos embrionados o porque el ensayo detecta ácido nucleico de virus no viables. Este hallazgo es importante y debe tenerse en cuenta al evaluar la detección del virus de desafío en estudios de vacunación y desafío, porque el método de qRT-PCR podría potencialmente identificar a aves positivas que de otro modo sería negativas mediante la detección del virus en huevos embrionados; lo que podría dar lugar a una evaluación más estricta de la eficacia de la vacuna. También se encontró que los iniciadores y las sondas diseñados de manera específica para el tipo del virus de bronquitis fueron en general menos sensibles que los iniciadores y sondas universales. Sólo el ensayo de específico para Ark- DPI en el SmartCycler II y los ensayos específicos para Ark- DPI, Mass41 y DE072/GA98 (para la detección del virus GA98 solamente) en el ABI 7500 fueron comparables en sensibilidad con la detección de virus en huevos embrionados. Se encontraron que una serie de variables, como el tipo de virus examinados, la eficiencia y estabilidad de los iniciadores y de las sondas, así como las condiciones del ensayo, incluyendo la plataforma del termociclador, pueden afectar los datos obtenidos con los ensayos de qRT-PCR. Estos resultados indican que los ensayos de qRT-PCR se pueden utilizar para detectar virus de desafío, pero cada ensayo, incluyendo las condiciones del ensayo y del termociclador, deben ser evaluadas de forma individual si se espera que los datos sean comparables a la detección de virus en huevos embrionados.

Evaluating Protection Against Infectious Bronchitis Virus by Clinical Signs, Ciliostasis, Challenge Virus Detection, and Histopathology

SUMMARY In this study, we examined the association among clinical signs, ciliostasis, virus detection, and histopathology for evaluating protection of vaccinated chickens against homologous and heterologous infectious bronchitis virus (IBV) challenge. At 5 days following challenge with IBV, we found a good correlation among clinical signs, ciliostasis in the trachea, challenge virus detection, and microscopic lesions in the trachea, with all four criteria being negative in fully protected birds and positive in fully susceptible birds. In partially protected birds we observed clinical signs and detected challenge virus; however, the ciliated epithelium was intact. In a second experiment, we challenged fully protected, partially protected, and fully susceptible birds with IBV, and then at 5 days postchallenge we gave the birds an opportunistic bacterium intranasally. Twenty Bordetella avium colonies were recovered from one of five fully protected birds, and only five colonies were isolated from two of five partially protected birds without ciliostasis, whereas in birds with ciliostasis, numerous colonies were isolated. Obviously, decreasing IBV infection and replication in the upper respiratory tract will decrease transmission and mutations, leading to variant viruses, and herein we demonstrate that protection of the cilia will decrease secondary bacterial infections, which have been shown to lead to condemnations and increased mortality. Thus, it appears that examining both criteria would be important when evaluating IBV vaccine efficacy.

Induced pluripotency in chicken embryonic fibroblast results in a germ cell fate.

Germ cells (GCs) are critically important as the vehicle that passes genetic information from one generation to the next. Correct development of these cells is essential and perturbation in their development often leads to reproductive failure and disease. Despite the importance of GCs, little is known about the mechanisms underlying the acquisition and maintenance of the GC character. Using a reprogramming strategy, we demonstrate that overexpression of ectopic transcription factors in embryonic fibroblasts can lead to the generation of chicken induced primordial germ cells (ciPGCs). These ciPGCs express pluripotent markers POU5F1, SSEA1, and the GC defining proteins, CVH and DAZL, closely resembling in vivo sourced PGCs instead of embryonic stem cells. Moreover, CXCR4 expressing ciPGCs were capable of migrating to the embryonic gonad after injection into the vasculature of stage 15 embryos, indicating the acquisition of a GC fate in these cells. Direct availability of ciPGCs in vitro would facilitate the study of GC development as well as provide a potential strategy for the conservation of important genetics of agricultural and endangered birds using somatic cells.

Expression of green fluorescent protein in the chicken using in vivo transfection of the piggyBac transposon

The chicken is a well-established model system for studying developmental biology and is recognized as one of the top food production animals in the world. For this reason the chicken is an excellent candidate for transgenic applications, as the technology can be applied to both areas of research. Transgenic technology has not been broadly utilized in the chicken model, however, primarily due to difficulties in targeting germ cells and establishing germ line transmission. Transgenic technologies using non-replicating viral particles have been used in the chick, but are unsuitable for many applications because of size and sequence restraints and low efficiency. To create a more versatile method to target chick germ line stem cells, we utilized the transposable element system piggyBac paired with an in vivo transfection reagent, JetPEI. piggyBac has been previously shown to be highly active in mammalian cells and will transpose into the chicken genome. Here, we show that JetPEI can transfect multiple chick cell types, most notably germline stem cells. We also show that pairing these two reagents is a viable and reproducible method for long-term expression of a transgene in the chicken. Stable expression of the green fluorescent protein (GFP) transgene was seen in multiple tissue types including heart, brain, liver, intestine, kidney and gonad. Combining an in vivo transfection strategy with the PB system provides a simple and flexible method for efficiently producing stable chimeric birds and could be used for production of germ line transgenics.

Nonviral Minicircle Generation of Induced Pluripotent Stem Cells Compatible with Production of Chimeric Chickens

Chickens are vitally important in numerous countries as a primary food source and a major component of economic development. Efforts have been made to produce transgenic birds through pluripotent stem cell [primordial germ cells and embryonic stem cells (ESCs)] approaches to create animals with improved traits, such as meat and egg production or even disease resistance. However, these cell types have significant limitations because they are hard to culture long term while maintaining developmental plasticity. Induced pluripotent stem cells (iPSCs) are a novel class of stem cells that have proven to be robust, leading to the successful development of transgenic mice, rats, quail, and pigs and may potentially overcome the limitations of previous pluripotent stem cell systems in chickens. In this study we generated chicken (c) iPSCs from fibroblast cells for the first time using a nonviral minicircle reprogramming approach. ciPSCs demonstrated stem cell morphology and expressed key stem cell markers, including alkaline phosphatase, POU5F1, SOX2, NANOG, and SSEA-1. These cells were capable of rapid growth and expressed high levels of telomerase. Late-passage ciPSCs transplanted into stage X embryos were successfully incorporated into tissues of all three germ layers, and the gonads demonstrated significant cellular plasticity. These cells provide an exciting new tool to create transgenic chickens with broad implications for agricultural and transgenic animal fields at large.

Polymorphisms in the S1 spike glycoprotein of Arkansas-type infectious bronchitis virus (IBV) show differential binding to host tissues and altered antigenicity

Abstract Sequencing avian infectious bronchitis virus spike genes re-isolated from vaccinated chicks revealed that many sequence changes are found on the S1 spike gene. In the ArkDPI strain, Y43H and ∆344 are the two most common changes observed. This study aims to examine the roles of Y43H and ∆344 in selection in vivo. Using recombinant ArkDPI S1 proteins, we conducted binding assays on chicken tracheas and embryonic chorioallantoic membrane (CAM). Protein histochemistry showed that the Y43H change allows for enhanced binding to trachea, whereas the ArkDPI S1 spike with H43 alone was able to bind CAM. Using Western blot under denaturing conditions, ArkDPI serotype-specific sera did not bind to S1 proteins with ∆344, suggesting that ∆344 alters antigenicity of S1. These findings are important because they propose that specific changes in S1 enhances virus fitness by more effective binding to host tissues (Y43H) and by evading a vaccine-induced antibody response (∆344).

Identification of Avian Coronavirus in Wild Aquatic Birds of the Central and Eastern USA

Abstract Coronaviruses (CoVs) are worldwide in distribution, highly infectious, and difficult to control because of their extensive genetic diversity, short generation time, and high mutation rates. Genetically diverse CoVs have been reported from wild aquatic birds that may represent a potential reservoir for avian CoVs as well as hosts for mutations and recombination events leading to new serotypes or genera. We tested 133 pooled samples representing 700 first-passage (in eggs) and 303 direct cloacal swab transport media samples from wild aquatic birds in the US that were avian influenza-negative. We isolated RNA from frozen samples and performed reverse transcriptase-PCR using a published universal CoV primer set. Of the samples tested, one from a Ruddy Turnstone (Arenaria interpres) was positive for CoV, showing nucleotide sequence similarity to a duck coronavirus (DK/CH/HN/ZZ2004). These data indicate a possible low prevalence of CoVs circulating in wild aquatic birds in the eastern half of the US.

Hatchery Spray Cabinet Administration Does Not Damage Avian Coronavirus Infectious Bronchitis Virus Vaccine Based on Analysis by Electron Microscopy and Virus Titration

SUMMARY Previous studies in our laboratory showed that the Arkansas–Delmarva Poultry Industry (Ark-DPI) vaccine given to 1-day-old chickens by hatchery spray cabinet replicated poorly and failed to adequately protect broilers against homologous virus challenge, whereas the same vaccine given by eye-drop did replicate and the birds were protected following homologous virus challenge. To determine if mechanical damage following spray application plays a role in failure of the Ark-DPI vaccine, we examined the morphology of three Ark-DPI vaccines from different manufacturers using an electron microscope and included a Massachusetts (Mass) vaccine as control. One of the Ark-DPI vaccines (vaccine A) and the Mass vaccine had significantly (P < 0.005) fewer spikes than the other two Ark-DPI vaccines. We also found that the Ark-DPI and Mass vaccines had significantly (P < 0.005) fewer spike proteins per virus particle when compared to their respective challenge viruses. This observation is interesting and may provide some insight into the mechanism behind infectious bronchitis virus attenuation. No obvious differences were observed in virus morphology and no consistent trend in the number of spikes per virion was found in before- and after-spray samples. We also determined the vaccine titer before and after spray in embryonated eggs and found that both Ark-DPI and Mass vaccines had a similar drop in titer, 0.40 log10 and 0.36 log10, respectively. Based on these data, it appears that mechanical damage to the Ark-DPI vaccine is not occurring when delivered by a hatchery spray cabinet, suggesting that some other factor is contributing to the failure of that vaccine when given by that method. RESUMEN La administración de la vacuna contra bronquitis infecciosa por aerosol con un gabinete en la planta de incubación no daña al coronavirus aviar determinado por microscopía electrónica y por titulación del virus. Estudios previos han demostrado que la vacuna Arkansas tipo Industria Avícola de Delmarva (Ark-DPI) aplicada a pollos de 1 día de edad por aspersión mediante un gabinete mostró una replicación pobre y no protegió adecuadamente a los pollos contra el desafío con un virus homólogo, mientras que la misma vacuna administrada por gota ocular, mostró replicación y las aves estuvieron protegidas después de la exposición al virus homólogo. Para determinar si ocurre daño mecánico después de la aplicación por aerosol y si este daño juega un papel en la falla de la vacuna Ark-DPI, se analizó la morfología de tres vacunas Ark-DPI de tres diferentes fabricantes utilizando un microscopio electrónico y se incluyó una vacuna serotipo Massachusetts como control. Una de las vacunas Ark-DPI (vacuna A) y la vacuna contra el serotipo Massachusetts mostraron significativamente (P <0.005) menos espículas que las otras dos vacunas Ark-DPI. También se encontró que las vacunas Ark-DPI y Massachusetts tuvieron significativamente (P <0.005) menos espículas por partícula viral en comparación con sus respectivos virus de desafío. Esta observación es interesante y puede proporcionar alguna información sobre el mecanismo de la atenuación del virus de la bronquitis infecciosa. No se observaron diferencias evidentes en la morfología del virus y no se encontró ninguna tendencia constante en el número de espigas por virión en muestras antes y después del aerosol. También se determinó el título de la vacuna por inoculación en huevos embrionados de pollo antes y después del aerosol y se encontró que ambas vacunas Ark-DPI y la vacuna Massachusetts mostraron una disminución similar en el título, 0.40 log10 y 0.36 log10, respectivamente. Con base en estos datos, parece que no ocurre daño mecánico a la vacuna Ark-DPI cuando se aplica mediante un gabinete de aspersión en la planta de incubación, lo que sugiere que algún otro factor está contribuyendo con la falla de la vacuna cuando se administra por este método.