Enhanced Protection by Recombinant Newcastle Disease Virus Expressing Infectious Bronchitis Virus Spike Ectodomain and Chicken Granulocyte-Macrophage Colony-Stimulating Factor.

We previously reported that recombinant Newcastle disease virus LaSota (rLS) expressing infectious bronchitis virus (IBV) Arkansas (Ark)-type trimeric spike (S) ectodomain (Se; rLS/ArkSe) provides suboptimal protection against IBV challenge. We have now developed rLS expressing chicken granulocyte-macrophage colony-stimulating factor (GMCSF) and IBV Ark Se in an attempt to enhance vaccine effectiveness. In the current study, we first compared protection conferred by vaccination with rLS/ArkSe and rLS/ArkSe.GMCSF. Vaccinated chickens were challenged with virulent Ark, and protection was determined by clinical signs, viral load, and tracheal histomorphometry. Results showed that coexpression of GMCSF and the Se from rLS significantly reduced tracheal viral load and tracheal lesions compared with chickens vaccinated with rLS/ArkSe. In a second experiment, we evaluated enhancement of cross-protection of a Massachusetts (Mass) attenuated vaccine by priming or boosting with rLS/ArkSe.GMCSF. Vaccinated chickens were challenged with Ark, and protection was evaluated. Results show that priming or boosting with the recombinant virus significantly increased cross-protection conferred by Mass against Ark virulent challenge. Greater reductions of viral loads in both trachea and lachrymal fluids were observed in chickens primed with rLS/ArkSe.GMCSF and boosted with Mass. Consistently, Ark Se antibody levels measured with recombinant Ark Se protein-coated ELISA plates 14 days after boost were significantly higher in these chickens. Unexpectedly, the inverse vaccination scheme, that is, priming with Mass and boosting with the recombinant vaccine, proved somewhat less effective. We concluded that a prime and boost strategy by using rLS/ArkSe.GMCSF and the worldwide ubiquitous Mass attenuated vaccine provides enhanced cross-protection. Thus, rLS/GMCSF coexpressing the Se of regionally relevant IBV serotypes could be used in combination with live Mass to protect against regionally circulating IBV variant strains.

Protección incrementada por el virus recombinante de la enfermedad de Newcastle que expresa el ectodominio de la espícula del virus de la bronquitis infecciosa y el factor estimulante de colonias de granulocitos y macrófagos del pollo. Anteriormente se reportó que la cepa LaSota recombinante del virus de la enfermedad de Newcastle (rLS) que expresa el ectodominio de la espícula trimérica (S) de tipo Arkansas (Ark) del virus de la bronquitis infecciosa (IBV) (Se; rLS/ArkSe) proporciona una protección subóptima contra la exposición al virus de la bronquitis infecciosa. Ahora se ha desarrollado hemos desarrollado una cepa LaSota recombinante (rLS) que expresa el factor estimulante de colonias de granulocitos y macrófagos de pollo (GMCSF) y la espícula del virus de bronquitis Arkansas en un intento para mejorar la efectividad de la vacuna. En el estudio actual, primero se comparó la protección conferida por la vacunación con los virus rLS/ArkSe y rLS/ArkSe.GMCSF. Los pollos vacunados se desafiaron con un virus Arkansas virulento y la protección se determinó mediante los signos clínicos, la carga viral y la histomorfometría de la tráquea. Los resultados mostraron que la coexpresión del factor estimulante de colonias de granulocitos y macrófagos de pollo y la espícula de la cepa recombinante LaSota redujo significativamente la carga viral traqueal y las lesiones traqueales en comparación con los pollos vacunados con el virus rLS/ArkSe. En un segundo experimento, se evaluó el incremento en la protección cruzada por una vacuna atenuada de Massachusetts (Mass) mediante la primovacunación o la vacunación de refuerzo con rLS/ArkSe.GMCSF. Los pollos vacunados fueron desafiados con el virus Arkansas y se evaluó la protección. Los resultados mostraron que la primovacunación o la vacunación de refuerzo con el virus recombinante aumentó significativamente la protección cruzada conferida por el virus Massachusetts contra el desafío virulento con el virus Arkansas. Se observaron mayores reducciones de las cargas virales en los fluidos traqueales y lagrimales en pollos primovacunadoss con rLS/ArkSe.GMCSF y con vacunación de refuerzo con Massachusetts. De manera consistente, los niveles de anticuerpos Ark Se medidos con placas de ELISA recubiertas con proteína Ark Se recombinante a los 14 días después del refuerzo fueron significativamente más altos en estos pollos. De manera inesperada, el esquema de vacunación inverso, es decir, la primovacunación con Massachusetts y el refuerzo con la vacuna recombinante, resultó menos efectivo. Se concluye que una estrategia de primovacunación y refuerzo mediante el uso de rLS/ArkSe.GMCSF y la vacuna atenuada con Massachusetts usada en todo el mundo proporciona una protección cruzada aumentada. Por tanto, el virus rLS/GMCSF que coexpresa la proteína de la espícula de los serotipos regionales relevantes de bronquitis infecciosa podría usarse en combinación con una vacuna viva Massachusetts para proteger contra cepas variantes del virus de la bronquitis infecciosa que circulan regionalmente.

Limited Protection Conferred by Recombinant Newcastle Disease Virus Expressing Infectious Bronchitis Spike Protein.

Recombinant Newcastle disease virus (NDV) LaSota (LS) expressing secreted trimeric spike (S)-ectodomain (Se) of infectious bronchitis virus (IBV) (rLS/IBV.Se) was developed and evaluated for protection conferred against IBV challenge. The IBV S-ectodomain protein, which is S excluding the transmembrane anchor and short cytoplasmic domain of S2, expressed from recombinant LS corresponds to an Arkansas (Ark)-type IBV. In a first experiment, chickens were primed at 1 day of age or primed at 1 day of age and boosted at 14 days of age with 104 50% embryo infectious doses (EID50)/bird of rLS/IBV.Se and challenged with a virulent Ark strain. A single vaccination proved completely ineffective at protecting chickens against challenge, whereas priming and boosting reduced clinical signs and tracheal lesions but did not reduce viral load in lachrymal fluids. In experiment 2, the vaccine dose was increased to 107 EID50/bird and a different virulent Ark strain was used for challenge. In addition, chickens were singly immunized on either day 1 or day 10 after hatch. NDV antibody levels detected in vaccinated chickens were moderate, with hemagglutination inhibition titers varying between 4 and 5 log2. Slightly higher antibody levels to NDV were observed in chickens vaccinated on day 10 versus day 1 but without the difference achieving statistical significance. In contrast, antibody responses measured using recombinant IBV S1 protein-coated ELISA plates were significantly greater in chickens vaccinated on day 10 than on day 1. The use of a higher rLS/IBV.Se dose substantially enhanced the success of a single vaccination compared to experiment 1. Signs and tracheal lesions were reduced more effectively in chickens vaccinated at day 10 after hatch. However, as in experiment 1, vaccination did not reduce the viral loads in tear fluids of challenged chickens. Similar results, in which no reduction in viral load in the trachea was apparent from rLS/IBV.S vaccination, have been obtained by others. Further work is needed to understand the immune responses induced by this recombinant virus that seems to provide some protection against the disease but does not reduce viral loads in the upper respiratory tract.

Protección limitada conferida por un virus recombinante de la enfermedad de Newcastle que expresa la proteína de la espícula del virus de la bronquitis infecciosa. Un virus de la enfermedad de Newcastle (NDV) LaSota (LS) recombinante que expresa el ectodominio de la proteína de la espícula (S) trimérica en forma secretoria (Se) del virus de la bronquitis infecciosa (IBV) (rLS/IBV.Se) fue desarrollado y evaluado para la protección contra el desafío con el virus de la bronquitis infecciosa. El ectodominio de la proteína S del virus de la bronquitis infecciosa, que consiste en la proteína S correspondiente a la cepa Arkansas, excluyendo su anclaje transmembranario y el dominio citoplasmático corto de la proteína S2 y expresada por una cepa LaSota recombinante. En un primer experimento, los pollos fueron primovacunados al primer día de edad, o primovacunados al primer día y con un refuerzo a los 14 días de edad con 104 dosis infecciosas de embriones de pollo 50% (EID50) por ave del virus recombinante rLS/IBV.Se y desafiados con un virus virulento cepa Arkansas. Una sola vacunación demostró ser completamente ineficaz para proteger a los pollos contra el desafío, mientras que la primovacunación y el refuerzo redujeron los signos clínicos y las lesiones traqueales, pero no redujeron la carga viral en los fluidos lagrimales. En el experimento 2, la dosis de la vacuna se incrementó a 107 EID50/ave y se usó una cepa Arkansas virulenta diferente para el desafío. Además, los pollos se inmunizaron individualmente al primer día de edad o al día 10 después de la eclosión. Los niveles de anticuerpos contra el virus de Newcastle detectados en pollos vacunados fueron moderados, con títulos de inhibición de la hemaglutinación que variaron entre 4 y 5 log2. Se observaron niveles de anticuerpos ligeramente superiores contra el virus de Newcastle en pollos vacunados el día 10 en comparación con los vacunados al primer día, pero sin que la diferencia alcanzara diferencia estadística significativa. En contraste, las respuestas de anticuerpos usando placas ELISA recubiertas con proteína recombinante S1 del virus de la bronquitis infecciosa fueron significativamente mayores en pollos vacunados en el día 10 en comparación con los pollos vacunados en el primer día. Con el uso de una dosis más alta del virus rLS/IBV.Se se aumentó sustancialmente el éxito de una vacunación única en comparación con el experimento 1. Los signos y las lesiones traqueales se redujeron de manera más efectiva en los pollos vacunados en el día 10 después de la eclosión. Sin embargo, como en el experimento 1, la vacunación no redujo las cargas virales en los fluidos lagrimales de los pollos desafiados. Resultados similares, en los que no se observó una reducción en la carga viral en la tráquea por la vacunación con rLS/IBV.Se se han obtenido por otros investigadores. Se requiere de más investigación para comprender las respuestas inmunes inducidas por este virus recombinante que parece proporcionar cierta protección contra la enfermedad, pero no reduce las cargas virales en el tracto respiratorio superior.

Infectious Bronchitis Virus Population Structure Defines Immune Response and Protection.

A commercial Arkansas (Ark) Delmarva Poultry Industry (DPI)-type vaccine and a more homogeneous population of that vaccine obtained previously through adaptation to chicken embryo kidney (CEK) cells (CEK-ArkDPI) were used as a model to further understand the impact of population genetic structure on generation of immune responses and protection. In a first experiment, vaccinated chickens were challenged with an IBV Ark99-type virulent strain (AL/4614/98). Despite extensive sequence similarity between the vaccines, the more heterogeneous commercial ArkDPI was more efficient at reducing viral loads in challenged chickens, while respiratory signs and tracheal lesions were reduced similarly by either vaccine. A distinct subpopulation of the Ark challenge virus showing asparagine at S1 position 56 was consistently negatively selected by immune pressure originating from vaccination with either vaccine. Antibody levels and antibody avidity to Ark-type S1 protein were greater in CEK-ArkDPI-vaccinated chickens compared to chickens vaccinated with the more diverse commercial ArkDPI vaccine. Synchronous replication of a homogeneous virus population likely elicits clonal expansion and affinity maturation of a greater number of responding B cells compared to a diverse virus population continuously changing its proportion of phenotypes during replication. The results of a second experiment showed that during initial vaccine virus replication (24 and 48 hr postvaccination), the virus population showing increased diversity (commercial ArkDPI) achieved higher concentrations of IBV RNA in the trachea compared to the more homogenous virus. mRNA expression of genes associated with innate immune responses in the trachea 48 hr postvaccination generally showed greater upregulation in chickens vaccinated with the heterogeneous commercial ArkDPI vaccine compared to the CEK-adapted virus. The greater upregulation of these genes is likely associated with higher virus replication achieved by the heterogeneous commercial vaccine. Thus, while the adaptive antibody response was favored by the more homogenous structure of the CEK-ArkDPI vaccine population (higher antibody levels and antibody avidity), the innate immune response was favored by the more diverse viral population of the commercial ArkDPI. We confirmed previous results that distinct subpopulations in wild Ark challenge virus become selected by immune pressure originating from vaccination, and we concluded that the population structure of IBV vaccines impacts innate immune response, antibody avidity, and protection.

La estructura poblacional del virus de la bronquitis infecciosa define la respuesta inmune y la protección. Se utilizaron una vacuna comercial del tipo Arkansas (Ark) Industria Avícola de Delmarva (Delmarva Poultry Industry: DPI) y una población más homogénea de esa vacuna obtenida previamente a través de su adaptación a las células de riñón embrionario de pollo (CEK) (CEK-ArkDPI) como modelos para comprender mejor el impacto de la estructura genética de la población en la generación de respuestas inmunes y protección. En un primer experimento, los pollos vacunados fueron desafiados con una cepa virulenta de tipo Ark99 del virus de la bronquitis infecciosa (AL/4614/98). A pesar de la extensa similitud en las secuencias entre las vacunas, la cepa comercial Arkansas DPI más heterogénea fue más eficiente en la reducción de las cargas virales en los pollos desafiados, mientras que los signos respiratorios y las lesiones traqueales se redujeron de manera similar con cualquiera de las dos vacunas. Una subpoblación distinta del virus de desafío Arkansas que muestra asparagina en la posición 56 de la proteína S1 fue seleccionada de forma negativa consistentemente por la presión inmunitaria originada por la vacunación con cualquiera de las dos vacunas. Los niveles de anticuerpos y su avidez hacia la proteína S1 tipo Arkansas fueron mayores en los pollos inmunizados con la vacuna CEK-ArkDPI en comparación con los pollos vacunados con la vacuna comercial Arkansas DPI más diversa. La replicación sincrónica de una población de virus homogénea probablemente provoca la expansión clonal y la maduración por afinidad de un mayor número de células B con capacidad de respuesta en comparación con una población de virus diversa que cambia continuamente su proporción de fenotipos durante la replicación. Los resultados de un segundo experimento mostraron que durante la replicación inicial del virus de la vacuna (24 y 48 horas después de la vacunación), la población de virus que mostró una mayor diversidad (Arkansas DPI comercial) alcanzó mayores concentraciones de ARN del virus de bronquitis infecciosa en la tráquea en comparación con el virus más homogéneo. La expresión de ARNm de genes asociados con respuestas inmunes innatas en la tráquea 48 h después de la vacunación generalmente mostró una mayor regulación positiva en pollos vacunados con la vacuna comercial heterogénea Arkansas DPI en comparación con el virus adaptado a células de riñón embrionario de pollo. Es probable que la mayor regulación positiva de estos genes esté asociada con una mayor replicación del virus lograda por la vacuna comercial heterogénea. Por lo tanto, mientras que la respuesta de anticuerpos adaptativos fue favorecida por la estructura más homogénea de la población de vacunas CEK-ArkDPI (mayores niveles de anticuerpos y mayor avidez de anticuerpos), la respuesta inmune innata fue favorecida por la población viral más diversa de la vacuna Arkansas DPI comercial. Se confirman resultados previos de que distintas subpoblaciones en el virus de desafío Arkansas de tipo silvestre se seleccionan por la presión inmune originada por la vacunación y se concluye que la estructura de la población de las vacunas contra el virus de la bronquitis infecciosa afecta la respuesta inmune innata, la avidez de los anticuerpos y la protección.

Infectious Bronchitis Virus Immune Responses in the Harderian Gland upon Initial Vaccination.

In recent years, Arkansas Delmarva Poultry Industry (ArkDPI)-derived infectious bronchitis (IB) virus (IBV) vaccines have been used to characterize the immune responses of chickens subsequent to vaccination on day of hatch or beyond. Perhaps because ArkDPI vaccines display increased heterogeneity, the results on cell immune responses have shown ambiguity. In the current study, we investigated the effects of vaccination with a highly stable and homogeneous Massachusetts (Mass)-type vaccine on days 1 or 7 of age on Harderian gland (HG) responses. Confirming previous studies, both IBV serum antibodies and lachrymal IgA levels were greater upon vaccination on day 7 compared with vaccination on day 1 of age. Unlike results with ArkDPI viruses, a clear trend was detected for both B and T cells in the HG after Mass-type vaccination. Consistent with antibody responses, B- and T-helper (CD3+CD4+) cell frequencies were higher in birds vaccinated on day 7 of age. Cytotoxic T cells (CD3+CD8+) were also increased compared with chickens vaccinated on day 1 of age. Depending on the most likely age of IB outbreaks to occur in a particular region, postponing the first IBV vaccination may optimize immune responses.

Nota de Investigación- Respuestas inmunes al virus de la bronquitis infecciosa en la glándula de Harder después de una vacunación inicial En los últimos años, las vacunas contra el virus de la bronquitis infecciosa (IB) derivadas de la cepa Arkansas Industria Avícola de Delmarva (ArkDPI) se han utilizado para caracterizar las respuestas inmunes de los pollos después de la vacunación en el día de la eclosión o en días posteriores. Posiblemente debido a que las vacunas ArkDPI muestran una mayor heterogeneidad, los resultados sobre las respuestas inmunes celulares han mostrado ambigüedad. En el presente estudio, se investigaron los efectos de la vacunación con una vacuna de tipo Massachusetts (Mass) que es altamente estable y homogénea en los días uno o siete de edad con relación a las respuestas en la glándula de Harder (HG). Confirmando estudios previos, tanto los anticuerpos séricos contra el virus de la bronquitis infecciosa como los niveles de IgA lacrimal fueron mayores tras la vacunación al día siete en comparación con la vacunación al primer día de edad. A diferencia de los resultados con los virus ArkDPI, se detectó una clara tendencia para las células B y T en la glándula de Harder después de la vacunación con el tipo Mass. De acuerdo con las respuestas de anticuerpos, las frecuencias de células B y T cooperadoras (CD3+ CD4+) fueron más altas en las aves vacunadas en el día siete de edad. Las células T citotóxicas (CD3+CD8+) también aumentaron en comparación con los pollos vacunados en el primer día de edad. Dependiendo de la edad más probable en que ocurran brotes por bronquitis infecciosa en una región en particular, posponer la primera vacuna contra el IBV puede optimizar las respuestas inmunes.

Early Vaccination of Chickens Induces Suboptimal Immunity Against Infectious Bronchitis Virus.

Infectious bronchitis virus (IBV) is highly prevalent in broiler chickens despite extensive vaccination commonly conducted early after hatch. The effects of early vaccination on immune responses were further investigated in chickens primed at increasing ages, followed by booster vaccination with an attenuated Arkansas (Ark) Delmarva Poultry Industry-type vaccine. Results show that vaccination on day 1 of age elicits significantly lower systemic and mucosal antibody responses compared with vaccination at later time points in the life of the chicken. The increase of IBV antibodies in serum from secondary responses after booster vaccination was more dramatic and significantly higher when measured by an Ark spike subunit 1 protein ELISA compared with measuring by non-Ark serotype whole-virus ELISA, which underlines the immunogenic importance of the virus spike at inducing antibodies. However, the levels achieved following boosting did not differ significantly between ages of priming. Thus, it seems that the booster vaccination mitigated the differences detected after prime immunization. In contrast to the continued rise of systemic antibodies after booster vaccination, the levels of mucosal IBV-specific immunoglobulin A decreased after booster vaccination. The recruitment or expansion of cluster of differentiation (CD)4+, CD8+, and CD4+/CD8+ T-cell populations in different immune effector sites was increased with age, but remained unaltered by IBV vaccination. In contrast, peripheral blood CD4+ cells showed a significant increase in IBV-vaccinated chickens compared with nonvaccinated age-matched controls both after primary and booster immunization. The results of the current study confirm that IBV vaccination on the day of hatch induces suboptimal IBV immune responses both in the systemic and mucosal compartments. This routine practice may be contributing to the immunologic escape of the virus and increased persistence of vaccine virus in vaccinated chickens. However, booster vaccination seems to overcome poor initial responses.

La vacunación temprana de pollos induce inmunidad subóptima contra el virus de la bronquitis infecciosa. El virus de la bronquitis infecciosa (IBV, por sus siglas en inglés) es altamente prevalente en los pollos de engorde a pesar de la vacunación extensiva que comúnmente se realiza temprano después de la eclosión. Los efectos de la vacunación temprana en las respuestas inmunitarias se investigaron más a fondo en pollos primovacunados a edades crecientes, seguidos de la vacunación de refuerzo con una vacuna atenuada tipo Arkansas (Ark) Delmarva Poultry Industry (Industria Avícola de Delmarva). Los resultados muestran que la vacunación en el día uno de edad provoca respuestas de anticuerpos sistémicos y de las mucosas significativamente menores en comparación con la vacunación en momentos posteriores en la vida del pollo. El aumento de los anticuerpos contra el virus de la bronquitis infecciosa en el suero en las respuestas secundarias después de la vacunación de refuerzo fue más marcado y significativamente mayor cuando se midió con un ensayo de inmunoabsorción con enzymas ligadas (ELISA) para la proteína de la subunidad 1 de la proteína de la espícula en comparación con la medición con un estuche de ELISA con el virus completo de serotipo diferente al Arkansas, lo que subraya la importancia inmunogénica de la espícula del virus en la inducción de anticuerpos. Sin embargo, los niveles alcanzados después del refuerzo no difirieron significativamente entre las edades de primovacunación. Por lo tanto, parece que la vacunación de refuerzo mitigó las diferencias detectadas después de la primo inmunización. En contraste con el aumento continuo de anticuerpos sistémicos después de la vacunación de refuerzo, los niveles de inmunoglobulina A específica para el virus de la bronquitis infecciosa en la mucosa disminuyeron después de la vacunación de refuerzo. El reclutamiento o la expansión de las poblaciones de células T CD4+, CD8+ y CD4+-CD8+ en diferentes sitios efectores inmunes se incrementó con la edad, sin embargo, permaneció inalterado por la vacunación contra el virus de la bronquitis infecciosa. En contraste, las células CD4+ de sangre periférica mostraron un aumento significativo en los pollos vacunados contra el virus de la bronquitis infecciosa en comparación con los controles no vacunados, tanto después de la primo inmunización como después de la inmunización de refuerzo. Los resultados de este estudio confirman que la vacunación contra el virus de la bronquitis infecciosa en el día de la eclosión induce respuestas inmunes subóptimas contra el mismo virus tanto en los compartimentos sistémicos como en los de las mucosas. Esta práctica de rutina puede contribuir al escape inmunológico del virus y al aumento de la persistencia del virus vacunal en pollos vacunados. Sin embargo, la vacunación de refuerzo parece mitigar las malas respuestas iniciales.

Infectious Bronchitis Virus Vaccination at Day 1 of Age Further Limits Cross Protection.

Cross-protection and immune responses elicited by infectious bronchitis virus (IBV) vaccination on Day 1 of age or at later time points were examined. Specific-pathogen-free chickens were vaccinated with a Massachusetts-type vaccine and heterologous challenge was performed with an Arkansas (Ark) -type virulent strain. In Trial 1, chickens vaccinated on Day 1 or Day 10 of age were challenged 21 days after vaccination. Analysis of tracheal histopathology and viral load demonstrated less cross protection when vaccination was performed on Day 1 of age. In Trial 2, chickens were vaccinated on Day 1 or Day 14 of age. A somewhat stronger systemic antibody response to IBV was detected in chickens vaccinated at 14 days of age. In addition, avidity of antibodies to Ark-type S1 protein elicited by vaccination at 14 days of age was greater. Differences in immune-cell populations in the Harderian gland (HG) observed at the time of sampling (35 days following vaccination) between chickens vaccinated at 1 day or 14 days of age indicated greater, rather than reduced, immune activity in the chickens vaccinated at 1 day of age. These differences are, perhaps, a result of the higher levels of persisting vaccine virus observed in the younger chickens. Both nonvaccinated/challenged groups showed significantly higher (P < 0.05) proportions of B cells and CD8+ T cells 7 days after challenge than age-matched vaccinated/challenged groups or age-matched nonvaccinated/nonchallenged control groups. These results indicate infiltration and/or expansion of B cells and CD8+ cells in HGs following challenge of nonvaccinated chickens. A fortuitous finding was that the more immature immune system of chickens vaccinated at 1 day of age was less effective at clearing vaccine virus after vaccination. Collectively, the current results indicate that IBV vaccination at 1 day of age can decrease the potential for heterologous cross protection compared with vaccination at least 10 days after hatch. A lower level of antibody affinity maturation likely contributes to decreased cross protection.

Límites en la protección cruzada inducida por la vacunación contra el virus de la bronquitis infecciosa en el primer día de edad. Se examinó la protección cruzada y la respuesta inmune inducida por la vacunación contra el virus de la bronquitis infecciosa en el primer día de edad o en posteriores puntos en el tiempo. Se vacunaron pollos libres de patógenos específicos con una vacuna de tipo Massachusetts y se realizó un desafío heterólogo con una cepa virulenta del tipo Arkansas (Ark). En el ensayo 1, pollos vacunados en el día uno o en el día 10 de edad fueron desafiados a los 21 días después de la vacunación. El análisis de la histopatología traqueal y de la carga viral demostró una menor protección cruzada cuando la vacunación se realizó en el día uno de edad. En el ensayo dos, los pollos fueron vacunados en el día uno o en el día 14 de edad. Se detectó una respuesta de anticuerpos sistémicos ligeramente más elevada contra el virus de la bronquitis infecciosa en los pollos vacunados a los 14 días de edad. Además, la avidez de los anticuerpos contra la proteína S1 tipo Arkansas inducida por la vacunación a los 14 días de edad fue mayor. Las diferencias en las poblaciones de células inmunitarias en la glándula de Harder observadas en el momento del muestreo 35 días después de la vacunación en los pollos vacunados al primer día o a los14 días de edad indicaron una mayor actividad inmunitaria en lugar de reducción en los pollos vacunados al primer día de edad, posiblemente como resultado de los niveles más altos de virus vacunal persistente observados en los pollos más jóvenes. Ambos grupos no vacunados y desafiados mostraron proporciones significativamente más altas (P <0.05) de células B y células T CD8+ siete días después del desafío en comparación con los grupos vacunados y desafiados de la misma edad o los grupos control no vacunados y no desafiados de la misma edad. Estos resultados indican la infiltración y/o expansión de las células B y de células CD8+ en la glándula de Harder después del desafío de los pollos no vacunados. Un hallazgo fortuito fue que el sistema inmune más inmaduro de los pollos vacunados al primer día de edad fue menos efectivo para eliminar el virus vacunal después de la vacunación. En conjunto, los resultados actuales indican que la vacunación con el virus de la bronquitis infecciosa al primer día de edad puede disminuir el potencial de protección cruzada heteróloga en comparación con la vacunación al menos 10 días después de la eclosión. Un nivel más bajo de maduración de la afinidad de anticuerpos probablemente contribuye a disminuir la protección cruzada.

Kidney Cell-Adapted Infectious Bronchitis ArkDPI Vaccine is Stable and Protective.

We previously demonstrated that adaptation of an embryo-attenuated infectious bronchitis virus (IBV) Arkansas (Ark) Delmarva Poultry Industry (DPI)-derived vaccine to chicken embryo kidney (CEK) cells (CEKp7) shifted the virus population towards homogeneity in spike (S) and nonstructural protein genes. Moreover, the typical Ark vaccine subpopulations emerging in chickens vaccinated with commercial Ark vaccines were not detected in chickens vaccinated with CEKp7, indicating that kidney-cell adaptation drastically increased the stability of the vaccine virus population in chickens. In the current study both conventional and next-generation sequencing results show that the changes achieved during CEK adaptation remained after five back passages in embryonated chicken egg (ECE). In a first protection study 1-day-old chickens were vaccinated with 104.0 or 105.0 50% embryo infectious doses (EID50)/chicken of the second ECE back passage of CEKp7 (CEKp7e2) and demonstrated protection against Ark virulent (106.0 EID50) challenge. In a second protection trial, protection by CEKp7e2 was compared with protection conferred by an attenuated commercial ArkDPI-derived vaccine different from that which the CEK-adapted virus originated. All vaccinated chicken groups showed a significant reduction of respiratory signs and viral load after Ark virulent challenge compared to unvaccinated-challenged controls. In CEKp7e2 vaccinated chickens viral subpopulations different from the challenge virus were detected after challenge in a marginal number (7%-8%) of chickens. In contrast, IBV S1 sequences that differed from the predominant population in the challenge virus were detected after challenge in a large number (77%) of chickens vaccinated with the commercial Ark attenuated vaccine. The CEK-adapted IBV ArkDPI-derived vaccine is a stable and effective vaccine, which drastically reduces the emergence of Ark-like viruses both at vaccination and after challenge.

Inactivation of Avian Influenza Virus in Nonpelleted Chicken Feed.

Corn stored outside could become contaminated with avian influenza virus (AIV) from wild bird droppings. AIV-contaminated ingredients could pass into the poultry flocks in nonpelleted chicken feed. The efficacy of two disinfectants at inactivating AIV in chicken feed was evaluated. Both Termin-8 (a blend of formaldehyde, propionic acid, terpenes, and surfactant) and Finio (a blend of approved phytochemicals and carboxylic acids) effectively inactivated AIV in chicken feed. Because stability of infectious AIV in chicken feed is limited, we evaluated addition of protein (skim milk powder) to the virus suspension. Protein prolonged the stability of AIV in untreated feed to 24 hr at 24 C. However, both feed disinfectants were able to inactivate the virus in feed even when protected by skim milk powder.

Kidney Cell-Adapted Infectious Bronchitis Virus Arkansas Delmarva Poultry Industry Vaccine Confers Effective Protection Against Challenge.

We previously demonstrated that adaptation of an embryo-attenuated infectious bronchitis virus (IBV) Arkansas Delmarva Poultry Industry (ArkDPI)-derived vaccine to chicken embryo kidney (CEK) cells shifted the virus population towards homogeneity in spike (S) and nonstructural protein genes. Moreover, the typical Ark vaccine subpopulations emerging in chickens vaccinated with commercial Ark vaccines were not detected in chickens vaccinated with the CEK-adapted virus. In this study, chickens vaccinated with a low dose (1.6 × 10(3) EID50/bird, where EID50 is 50% embryo infectious dose) of CEK-adapted Ark vaccine at 5 days of age showed a significant reduction of IBV RNA in lachrymal fluids and decreased incidence of IBV RNA detection in tracheal swabs 5 days after challenge compared to unvaccinated challenged chickens. In a second experiment, 5-day-old chickens were vaccinated with 10(4) or 10(5) EID50/chicken of CEK-adapted Ark vaccine, and protection was compared to chickens vaccinated with 10(5) EID50/chicken of the commercial ArkDPI-derived vaccine from which the CEK-adapted virus originated. All vaccinated chicken groups showed a significant reduction of respiratory signs and viral load 5 days after Ark virulent challenge compared to unvaccinated challenged controls. No viral subpopulations different from the challenge virus were detected in chickens vaccinated with CEK-Ark after challenge. In contrast, IBV S1 sequences differing from the predominant population in the challenge virus were detected in several chickens vaccinated with the commercial Ark attenuated vaccine. From an applied perspective, the CEK-adapted IBV ArkDPI-derived vaccine is an improved and effective vaccine candidate with which to protect chickens against virulent Ark-type strains.

Variability Assessment of California Infectious Bronchitis Virus Variants.

On the basis of the data from the California Animal Health and Food Safety Laboratory System, 1444 infectious bronchitis (IB) cases were diagnosed between 1997 and 2012. Epidemiologic analyses demonstrated two major IB virus (IBV) outbreak peaks, affecting mainly 35-to-49-day-old broiler chickens. California variant 1737 (CA1737) and California variant 1999 (Cal 99) IBV types were the most prevalent genotypes during the analyzed period. To further understand the increased prevalence of these genotypes, we assessed and compared the variability of the S1 gene hypervariable region of CA1737 and Cal 99 with the variability of IBV strains belonging to the Massachusetts 41 (M41) and Arkansas (Ark) types during serial passages in embryonated chicken eggs. On the basis of the S1 nonsynonymous changes, seven different subpopulations were detected in M41. However, the predominant population of the field strain M41 before passages continued to be predominant throughout the experiment. In contrast, Ark passaging resulted in the detection of 13 different subpopulations, and the field sequence became extinct after the first passage. In IBV Cal 99, eight different subpopulations were detected; one of these became predominant after the second passage. In CA1737, 10 different subpopulations were detected. The field strain major sequence was not detected after the first passage but reappeared after the second passage and remained at low levels throughout the experiment. Compared with M41 and Ark, Cal 99 and CA1737 showed intermediate variability.

Cross-Protection by Infectious Bronchitis Viruses Under Controlled Experimental Conditions.

Infectious bronchitis virus (IBV) cross-protection trials were performed in healthy chickens maintained under controlled environmental conditions. Chickens primed or primed and boosted with a Massachusetts (Mass)-type attenuated vaccine were subsequently challenged with either IBV Arkansas (Ark) or GA13-type virulent strains. In addition, Ark-vaccinated chickens were challenged with IBV GA13. Spike protein 1 (S1) amino acid identities between IBV vaccine and challenge strains varied from 76.0% to 77.3%. Contrary to expectations, assessments of clinical signs, viral load, and histopathology indicated a significant level of cross-protection among these antigenically distant IBV strains. Moreover, prime and booster vaccination with Mass protected against GA13 and improved protection against Ark when compared with Mass single vaccination. These results emphasize the need to include both single vaccination control groups and control groups primed and boosted with a single serotype when testing the efficacy of IBV protectotypes and/or novel IBV vaccine combinations against heterologous serotypes under controlled experimental conditions. Such controls are of distinct importance in experiments supporting the introduction of attenuated IBV vaccine strains exotic to regions, since these exotic strains may provide new genetic material for recombination and emergence of novel IBV strains.

Effects of Adaptation of Infectious Bronchitis Virus Arkansas Attenuated Vaccine to Embryonic Kidney Cells.

The population structure of an embryo-attenuated infectious bronchitis virus (IBV) Arkansas (Ark) Delmarva Poultry Industry (DPI)-derived vaccine was characterized during serial passages in chicken embryo kidney (CEK) cells and after back-passage in embryonated chicken eggs (ECE) and in chickens. Both conventional and deep-sequencing results consistently showed population changes occurred during adaptation to CEK cells. Specifically, 13 amino acid (aa) positions seemed to be targets of selection when comparing the vaccine genome prior to and after seven passages in CEK (CEKp7). Amino acid changes occurred at four positions in the spike (S) gene and, at two positions in the S gene, large shifts in frequencies of aa encoding were observed. CEK adaptation shifted the virus population towards homogeneity in S. The changes achieved in the S1 gene in CEKp7 were maintained after a back-passage in ECE. Outside the S gene, aa changes at three positions and large shifts in frequencies at four positions were observed. Synonymous nucleotide changes and changes in noncoding regions of the genome were observed at eight genome positions. Inoculation of early CEK passages into chickens induced higher antibody levels and CEKp4 induced increased respiratory signs compared to CEKp7. From an applied perspective, the fact that CEK adaptation of embryo-attenuated Ark vaccines reduces population heterogeneity, and that changes do not revert after one replication cycle in ECE or in chickens, provides an opportunity to improve commercial ArkDPI-derived vaccines.

S1 of distinct IBV population expressed from recombinant adenovirus confers protection against challenge.

Protective properties of three distinct infectious bronchitis virus (IBV) Ark Delmarva poultry industry (ArkDPI) S1 proteins encoded from replication-defective recombinant adenovirus vectors were investigated. Using a suboptimal dose of each recombinant virus, we demonstrated that IBV S1 amino acid sequences showing > or = 95.8% amino acid identity to the S1 of the challenge strain differed in their ability at conferring protection. Indeed, the S1 sequence of the IBV population previously designated C4 (AdIBVS1.C4), which protected the most poorly, differs from the S1 sequence of population C2 (AdIBVS1.C2), which provided the highest protection, only at amino acid position 56. The fact that a change in one amino acid in this region significantly altered the induction of a protective immune response against this protein provides evidence that the first portion of S1 displays relevant immunoprotective epitopes. Use of an optimal dose of AdIBVS1.C2 effectively protected chickens from clinical signs and significantly reduced viral load after IBV Ark virulent challenge. Moreover, increased numbers of both IgA and IgG IBV-specific antibody secreting lymphocytes were detected in the spleen after challenge. The increased response detected for both IgA and IgG lymphocytes after challenge might be explained by vaccine-induced B memory cells. The fact that a single vaccination with Ad/IBVS1.C2 provides protection against IBV challenge is promising, because Ad-vectored vaccines can be mass delivered by in ovo inoculation using automated in ovo injectors.

Conjunctiva-associated lymphoid tissue in avian mucosal immunity.

Conjunctiva-associated lymphoid tissue's (CALT) role in generating avian mucosal adaptive immunity was measured by analyzing cellular composition, expression of the polymeric immunoglobulin receptor (pIgR), and production of cytokines and antibodies in chickens ocular exposed to a replication-deficient adenovirus of serotype 5 (Ad5). These studies demonstrate that CALT contains B cells, γδ T cells, T helper, and cytotoxic T cells, and a T lymphocyte composition, which more resembles Harderian glands than spleen. CALT-derived lymphocytes contain antigen-specific, IgA-secreting plasma cells and cytokine-producing lymphocytes after ocular Ad5 vaccination. The expression of the pIgR in the CALT's lymphoepithelium emphasizes the importance of mucosal immune protection by paraocular lymphoid tissues. The CALT immune response after ocular Ad5 boosting was influenced by prior high dose in ovo Ad5 priming. Thus, both mucosal and systemic immunization influenced Ad5-induced IFN-γ responses in CALT.

Avian influenza adenovirus-vectored in ovo vaccination: target embryo tissues and combination with Marek's disease vaccine.

We investigated embryo tissues targeted by replication competent adenovirus (Ad)-free recombinant Ad expressing a codon-optimized avian influenza (AI) H5 gene from A/turkey/WI/68 (AdH5) when injected into 18-day embryonated eggs. We also evaluated the effects of concurrent in ovo vaccination with the experimental AdH5 vaccine and commercially available Marek's disease virus (MDV) vaccine combinations Rispens/turkey herpesvirus (HVT) or HVT/SB-1. Computed tomography indicates that in ovo injection on day 18 of incubation places the solution in the amnion cavity, allantoic cavity, or both. Ad DNA was consistently detected in the chorioallantoic membranes as well as in the embryonic bursa of Fabricius, esophagus, and thymus 3 days postinoculation. H5 expression in these tissues also was detected by immunofluorescence assay. These results indicate possible swallowing of vaccine virus contained in the amnion. In contrast, vaccine localization in the allantoic fluid would have allowed bursal exposure through the cloaca. When the AdH5 vaccine was used in combination with MDV, chickens responding to the AdH5 vaccine had similar AI antibody levels compared with AdH5-only-vaccinated birds. However, combined vaccinated groups showed reduced vaccine coverage to AI, suggesting some level of interference. The combination of AdH5 with MDV Rispens/HVT affected the vaccine coverage to AI more severely. This result suggests that the replication rate of the more aggressive Rispens strain of serotype 1 may have interfered with the Ad-vectored vaccine. Increasing the Ad concentration produced similar AI antibody titers and AI vaccine coverage when applied alone or in combination with the HVT/SB-1 vaccine. Ad DNA was detected in hatched chickens 2 days after hatch but was undetectable on day 9 after hatch. MDV DNA was detected in feather follicles of all vaccinated birds at 12 days of age. Thus, Ad-vector vaccination does not interfere with the efficacy of MDV vaccination by using any of the commonly used vaccine strains.

Posición de las escuelas de medicina colombianas a partir del ranking iberomaericano SIR-2010 / Construction of a Colombian scale for medical schools with the SIR 2010

Antecedentes. A la fecha no existe en Colombia forma aceptable de conocer una posición meritoria de las escuelas de medicina. Existen diversos parámetros que pueden permitir establecer un ranking. Se ha escogido para este artículo la clasificación emitida por el SIR 2010 como una aproximación inicial al tema. Objetivo. Establecer la posición de las escuelas de medicina colombianas a partir del ranking Iberoamericano SIR 2010. Material y métodos. Se utilizó el Ranking Iberoamericano SIR 2010 Ciencias de la Salud publicado por Scimago Institutions Rankings en el presente año, que utiliza los siguientes parámetros: producción científica, colaboración internacional, calidad científica promedio y porcentaje de publicaciones en revistas del primer cuartil SJR. Resultados. Según los parámetros adoptados por SIR 2010 las primeras diez instituciones educativas colombianas en el área de la medicina son: Universidad de Antioquia, Universidad Nacional de Colombia, Universidad del Valle, Pontificia Universidad Javeriana, Universidad Industrial de Santander, Universidad de Rosario, Universidad de Los Andes, Universidad CES, Universidad Pontificia Bolivariana y Universidad de Caldas. Conclusión. Es importante establecer parámetros académicos e investigativos que permitan posicionar a las escuelas de medicina en Colombia.

La enfermería en salud pública: el caso de la Influenza / The public health nursing: the case of influenza

Ante un brote de una enfermedad infecto-contagiosa, el estudio de contactos limita la transmisión de esta. El objetivo de este trabajo fue identificar a los contactos de los posibles casos de influenza en trabajadores de la UNAM para establecer una comunicación y proporcionar educación para la salud sobre medidas higiénicas. Material y métodos: Se elaboró un cuestionario, se estableció contacto con los casos por vía telefónica y se llevó a cabo una visita domiciliaria. Se llevó a cabo un análisis descriptivo de los datos y una descripción de las experiencias y percepciones durante las visitas. Resultados: Se identificó que la mayoría de los contactos eran familiares directos de los casos, que no contaban con un esquema de vacunación completo, ni contra la influenza y la frecuencia de síntomas varió de 1 hasta 4. Comentarios finales: Ante una situación de este tipo (la Pandemia del Virus de la Influenza Humana A (H1N1)) el papel de la enfermera en salud pública es de suma importancia no solo en la búsqueda de los casos y sus contactos, sino también en la orientación y educación de la población en relación a las medidas preventivas.

In the view of an infectious and contagious disease epidemic, the identification of the contacts limits its transmission. The objective of this work was to identify the contacts of the possible cases of UNAM workers in order to establish communication and provide health education about hygiene measures. Material and methods: A questionnaire was prepared, telephone contact was established with the cases, and a home visit was arranged. A descriptive analysis of the data was carried out, as well as, a description of the experiences and perception during the home visit. Results: Most identified contacts were relatives of the cases, which did not have a complete vaccine scheme, no even against influenza, and the frequency of symptoms varied between 1 a 4. Final comments: In a situation like this (A(H1N1) human influenza virus pandemic) the public health nurse roll is of great importance, not just in the identification of the cases and its contacts, but also in the orientation and education of the population in relation to preventive measures.

Pathogenicity of infectious bursal disease virus variant AL2 in young chickens.

Limited information is available on the effects of the recently emerged infectious bursal disease virus (IBDV) variant AL2. In this study, the effects of inoculation of 4-day-old chickens with increasing doses of IBDV AL2 were characterized. IBDV AL2 induced neither overt clinical signs nor mortality. Infected chickens showed reduced bursa indices (BI) and bursa lymphocytic depletion, as determined by histomorphometry. However, histomorphometry and BI values differed during the early stages of the infection. Because data from bursa histomorphometry were consistent with viral RNA detection, such values seem to be more appropriate for the assessment of AL2 viral infectivity in chickens. Both the histomorphometry and BI data indicated a dose-effect pattern. However, with time, even low doses of the virus ultimately resulted in significant damage to the bursa. Samples of spleen were used to assess B- (IgM+) and T- (CD4+ and CD8+) cell populations by flow cytometry. Infected chickens showed a significant increase of splenic IgM+ cells at 5 and 8 days postinoculation (PI). On day 8 PI, the number of total IgM+ cells in the spleen was inversely related to the virus concentration. Others have shown that cell-mediated immunity is essential for protection against IBDV. Our results indicate a significant increase (P < 0.05) of total spleen CD4+ cell counts on day 8 PI in birds that received higher virus concentrations, indicating a role for these cells in protective immunity, while CD8 cell counts remained unchanged. We speculate that the changes in splenic CD4+ and IgM+ cell populations are associated with protective immune responses against IBDV in the host.

Effects of chicken anemia virus and infectious bursal disease virus in commercial chickens.

The effects of chicken anemia virus (CAV) and infectious bursal disease virus (IBDV) coinfection in commercial layer-type and meat-type (broiler) chickens with specific maternal immunity were evaluated. In addition, the broiler progeny used had been vaccinated in ovo against IBDV. Layer chickens were inoculated intramuscularly on day 3 of age with CAV and orally on day 7 of age with an IBDV standard strain (APHIS). Broiler chickens were exposed to CAV and/or an IBDV variant strain (AL2) via the drinking water on days 3 and 14 of age. Following CAV and IBDV inoculation neither mortality nor overt clinical disease was observed in any layer or broiler group. In spite of maternal immunity against both IBDV and CAV, mean hematocrits of all layer groups inoculated with CAV (CAV, CAV + APHIS) were lower than uninfected chickens. IBDV APHIS alone or in combination with CAV did not affect the layer weight gain. However, on day 30 of age and concomitantly with maternal antibody decay, bursa lymphocyte depletion became evident in CAV + APHIS-infected layer chickens. These birds (CAV + APHIS) also seroconverted to IBDV on day 35 of age. CAV persisted at low levels in the layer chickens throughout the experimental period in CAV- and CAV+APHIS-infected chickens. Similarly, infected broiler chickens did not show changes in weight gain. Compared to CAV-infected or uninfected controls, CAV+AL2- and AL2-infected broiler chickens showed significant lymphocyte depletion in the bursa as assessed both by bursal indices and histomorphometry. Broilers also seroconverted to IBDV after day 30 of age confirming that bursal lymphocyte depletion was due to IBDV resuming replication. Thymus histomorphometry revealed significant lymphocyte depletion in all infected broiler groups at 30 days of age, but only in CAV+AL2-infected broiler chickens at 41 days of age, suggesting that IBDV infection delayed repopulation of the thymus.

Protection of chickens against avian influenza with nonreplicating adenovirus-vectored vaccine.

Protective immunity against avian influenza (AI) virus has been elicited in chickens by single-dose in ovo or i.m. vaccination with a replication-competent adenovirus (Ad)-free human Ad vector encoding the AI virus A/Turkey/Wisconsin/68 H5 (AdTW68. H5) or the A/Chicken/New York/94 H7 (AdChNY94. H7) hemagglutinin (HA). The AdTW68.H5-vaccinated chickens were protected against both H5N1 and H5N2 highly pathogenic AI virus challenges. The AdChNY94. H7-vaccinated chickens were protected against an H7N3 highly pathogenic avian influenza virus challenge. Chickens vaccinated in ovo with AdTW68.H5 followed by posthatch i.m. vaccination with AdChNY94.H7 responded to both vaccinations, with robust antibody titers against both the H5 and H7 AI proteins. The use of a synthetic AI H5 HA gene codon optimized to match the tRNA pool found in chicken cells is more potent than the cognate H5 HA gene. Mass administration of this AI vaccine can be streamlined with available robotic in ovo injectors. In addition, Ad5-vectored vaccines can be produced rapidly and the safety margin of the nonreplicating vector is superior to that of a replicating counterpart. Furthermore, this mode of vaccination will not interfere with epidemiological surveys of natural AI infections. Finally, the demonstration that Ad-vectored vaccines can be administered repeatedly without appreciably losing potency highlights the commercial potential of this new class of vaccine in poultry.