In ovo vaccination with herpesvirus of turkey enhances innate and cellular responses in meat-type chickens: Effect of vaccine dose and strain.

In ovo vaccination with herpesvirus of turkey (HVT) or recombinant HVT (rHVT) is commonly used in meat-type chickens. Previous studies showed that in ovo vaccination with HVT enhances innate, cellular, and humoral immune responses in egg-type chicken embryos. This study evaluated if in ovo vaccination with HVT hastens immunocompetence of commercial meat-type chickens and optimized vaccination variables (dose and strain of HVT) to accelerate immunocompetence. A conventional HVT vaccine was given at recommended dose (RD), HVT-RD = 6080 plaque forming units (PFU), double-dose (2x), half-dose (1/2), or quarter-dose (1/4). Two rHVTs were given at RD: rHVT-A = 7380 PFU, rHVT-B = 8993 PFU. Most, if not all, treatments enhanced splenic lymphoproliferation with Concanavalin A and increased the percentage of granulocytes at day of age. Dose had an effect and HVT-RD was ideal. An increase of wing-web thickness after exposure to phytohemagglutinin-L was only detected after vaccination with HVT-RD. Furthermore, compared to sham-inoculated chickens, chickens in the HVT-RD had an increased percentage of CD3+ T cells and CD4+ T-helper cells, and increased expression of major histocompatibility complex (MHC)-II on most cell subsets (CD45+ cells, non-T leukocytes, T cells and the CD8+ and T cell receptor γδ T-cell subsets). Other treatments (HVT-1/2 and rHVT-B) share some of these features but differences were not as remarkable as in the HVT-RD group. Expression of MHC-I was reduced, compared to sham-inoculated chickens, in most of the cell phenotypes evaluated in the HVT-RD, HVT-2x and rHVT-A groups, while no effect was observed in other treatments. The effect of in ovo HVT on humoral immune responses (antibody responses to keyhole limpet hemocyanin and to a live infectious bronchitis/Newcastle disease vaccine) was minimal. Our study demonstrates in ovo vaccination with HVT in meat-type chickens can accelerate innate and adaptive immunity and we could optimize such effect by modifying the vaccine dose.

Biological and phylogenetic characterization of a genotype VII Newcastle disease virus from Venezuela: efficacy of field vaccination.

Here we report the biological and molecular characterization of a virulent genotype VII Newcastle disease virus (NDV) circulating in Venezuela and the assessment of the vaccination efficacy under field conditions compared to controlled rearing conditions. Biological pathotyping showed a mean embryo dead time of 50 h and an intracerebral pathogenicity index of 1.86. Sequence-based phylogenetic analysis demonstrated that the virus belongs to genotype VII in class II (a genotype often found in Asia and Africa), representing the first report of the presence of this genotype in the continent of South America. A vaccine-challenge trial in commercial broilers reared in fields or in a experimental setting included dual (live/killed) priming of 1-day-old chicks plus two live NDV and infectious bursal disease virus (IBDV) field vaccinations at days 7 and 17, followed by a very stringent genotype VII NDV challenge at day 28. Serology for NDV and IBDV, bursal integrity, and protection against NDV lethal challenge were assessed. At 28 days, field vaccinates showed significantly lower NDV (1,356 versus 2,384) and higher IBD (7,295 versus 1,489) enzyme-linked immunosorbent assay (ELISA) antibody titers than the experimentally reared birds. A lower bursal size and bursa-body weight ratio (P < 0.05) and higher bursa lesion score were also detected in the field set. Only 57.1% of field vaccinates survived the lethal challenge, differing (P < 0.05) from 90.5% survival in the experimental farm. Overall, results confirmed the presence of the genotype VII viruses in South America and suggest that field-associated factors such as immunosuppression compromise the efficacy of the vaccination protocols implemented.

Concomitant turkey herpesvirus-infectious bursal disease vector vaccine and oil-adjuvanted inactivated Newcastle disease vaccine administration: consequences for vaccine intake and protection.

Hatchery vaccination protocols in day-old chicks are designed to provide early priming and protection against several poultry diseases including, but not limited to, Marek's disease (MD), infectious bursal disease (IBD), and Newcastle disease (ND). The constraint of concomitant administration of live MD and IBD vaccines plus ND inactivated oil-adjuvanted vaccines (IOAVs) requires improvements in vaccine technology. Single-needle concomitant subcutaneous (SC) application of IBD/MDV and killed NDV vaccine and the use of viral vectors for expression of immunogenic proteins are a current trend in the industry. The objective of this work was to assess the compatibility of a turkey herpesvirus (HVT)-infectious bursal disease (vHVT-IBD) vector vaccine applied simultaneously with IOAV and to evaluate the consequences for vaccine intake, the need for additional immunizations with the respective vaccines, and protection. Five separate trials were performed using double- and/or single-needle injectors. The levels and persistence of vaccine intake, serologic response, vHVT-IBD virus combination with the MD Rispens strain, and/or live NDV vaccination were also assessed. Histopathology and PCR at injection sites showed adequate vaccine intake detected up to 44 days postvaccination. Serologic evidence of vaccine priming was observed, and all vaccinated groups differed (P < 0.05) from the control at different time points. MD, NDV, and IBD protection results after concomitant double-shot single-needle vaccination were near 85%, 95%, and 100%, respectively. Taken together the results indicate no deleterious effects on the efficacy of the vHVT-IBD vaccine monitored by vaccine intake, serologic and challenge results, and combinations after concomitant live/killed vaccination, suggesting the suitability of its use in hatchery vaccination. All types of injectors used as well as injection techniques, vaccines injected separately or together, gave the same results.

Eficacia de un herpesvirus de pavo recombinante expresando la proteína VP2 del virus de la enfermedad de Gumboro ante un desafío experimental / Efficacy of a Recombinant Turkey Herpesvirus Expressing the Viral Protein 2 of the Gumboro Disease Virus Against an Experimental Challenge

La vacunación temprana contra el virus de la enfermedad infecciosa de la bolsa (por sus siglas en inglés IBDV) es una práctica muy común; sin embargo, el uso de vacunas de baja atenuación puede comprometer la integridad de la bolsa de Fabricio en aves jóvenes generando inmunosupresión y el fracaso de los planes de vacunación. Una alternativa es la utilización de vectores virales para la expresión transgénica de proteínas inmunogénicas que pueden proporcionar protección adecuada sin el potencial daño a la bolsa. El objetivo del presente trabajo fue evaluar la protección contra un desafío experimental con cepas clásicas conferida por la vacunación al día de edad con VAXXITEK®, un herpesvirus de pavo (por sus siglas en inglés HVT) expresando la proteína inmunogénica VP2 de una cepa clásica del IBDV. Aves libres de patógenos específicos fueron vacunadas al día de edad por la vía subcutánea y luego desafiadas a los 18 ó 28 días de edad con la cepa STC del virus de la enfermedad de Gumboro. El criterio de protección incluyó signos clínicos, índice peso bolsa/peso corporal y la histopatología de la bolsa. En las aves vacunadas con VAXXITEK®, no se observaron signos clínicos o lesiones asociadas al desafío con la cepa STC, ni en el desafío temprano ni en el tardío. El índice bursal resultó significativamente menor en las aves no vacunadas que fueron desafiadas. Estos resultados indican que una dosis de la vacuna HVT-IBDV recombinante protege a las aves contra un desafío con cepas clásicas.

Early vaccination against infectious bursal disease virus (IBDV) is a common practice; however, the use of live attenuated vaccines may sometimes compromise the bursal integrity in young birds generating immunosupression and failures in vaccination programs. An alternative is the use of viral vectors for transgenic expression of immunogenic proteins that can provide adequate protection without the potential bursal damage. The objective of this work was to assess the protection against a classical strain challenge conferred by day-one vaccination using VAXXITEK®, a recombinant herpesvirus of turkey expressing the immunogenic viral protein 2 from a classical IBDV. Specific pathogen free (SPF) one-day old birds were vaccinated by the subcutaneous route and challenged with the STC IBDV strain at 18 or 28 days of age. The protection criteria included: clinical signs, bursa/bodyweight ratio and bursal histopathology. No clinical signs or STC challenge related bursal lesions were observed in the VAXXITEK® vaccinated birds at both early and late challenge. The bursal index was significantly lower in the unvaccinated challenged birds. These results indicate that single dose recombinant HVT-IBDV vaccination protects chickens against a classical strain challenge.

Detección y caracterización molecular de cepas variantes del virus de la enfermedad infecciosa de la bolsa en el Estado Zulia, Venezuela / Molecular detection and characterization of infectious bursal disease virus variant strains from Zulia State, Venezuela

Las cepas clásicas del virus de la enfermedad infecciosa de la bolsa (por sus siglas en inglés IBDV) están presentes en la industria avícola venezolana desde el último tercio del siglo pasado, a pesar de la implementación de programas intensivos de vacunación. Recientemente, se ha reportado la presencia de cepas variantes del IBDV en varios países de Latinoamérica. El presente trabajo reporta la identificación, mediante técnicas moleculares, de cepas variantes en granjas avícolas venezolanas. En parvadas de pollos de engorde de cuatro semanas de edad se tomaron bolsas de Fabricio e improntas en la tarjeta Whatman Indicadora Clásica FTA® para evaluación histopatológica y detección molecular, respectivamente. Para la caracterización molecular se utilizó la prueba de reacción en cadena por la polimerasa-transcriptasa reversa (por sus siglas en inglés RT-PCR) acoplada a secuenciación directa de nucleótidos. El peso vivo del ave, el índice peso bolsa/peso corporal, así como el peso y diámetro de la bolsa se consideraron como indicadores de inmunocompetencia a la cuarta semana. Todas las aves muestreadas (n=113) resultaron positivas a IBDV. Los virus provenientes de 10 de las 13 granjas mostraron alta similitud con cepas variantes (A y E). En concordancia con los resultados de RT-PCR, los hallazgos histopatológicos mostraron lesiones relacionadas con IBDV. Los indicadores morfológicos de inmunocompetencia se afectaron significativamente en las granjas donde se detectaron cepas variantes. En Venezuela no se utiliza vacuna viva contra cepas variantes, lo que incrementa el riesgo de inmunosupresión, fallas en los programas de vacunación y la susceptibilidad a enfermedades endémicas.

Classical infectious bursa disease virus (IBDV) has been present in the Venezuelan poultry industry since the last quarter of the past century despite intensive vaccination programs applied to control the disease. Lately, the presence of variant strains has been reported in several Latin-American countries. This work reports the molecular identification of variant IBDV strains in poultry farms from Venezuela. Bursal imprints in Whatman classic indicator FTA® cards and bursa tissues for histopathological analysis were collected from 4-week old broiler flocks. Reverse transcriptase polymerase chain reaction (RT-PCR) and direct nucleotide sequence were used for the virus molecular characterization. The bird´s body weight, the bursal index, the bursa weight and diameter were used to assess the immune status at four weeks of age. All the birds sampled (n =113) were IBDV positive. Viruses from 10 out of 13 farms showed high similarity with the IBDV variant strains (variants A and E). Histopathological findings where consistent with the RT-PCR, results showing IBDV related bursa damage in the infected birds. The immune status indicators were significantly affected in the farms where variant strains were detected. No variant strain live vaccination is currently used in Venezuela, increasing the risk of immunossupression, vaccine failure and susceptibility to endemic diseases.

Bursal restoration after intermediate and intermediate plus infectious bursal disease virus vaccination

Infectious bursal disease (IBD) is an acute, contagious, viral disease of young chickens characterized by diarrhea, ventpicking, trembling, incoordination, inflammation followed by atrophy of the bursa of Fabricius and by variable degrees of immunosuppression. The diseases is caused by the infectious bursal disease virus (IBDV) which upon its antigenic characteristics and pathogenicity has been classified as classic (mild, intermediate and intermediate plus) strains, very virulent IBDV (vvIBDV) and variant strains. With the widespread presence of vvIBDV, the poultry industry has resorted to the use of less attenuated vaccines raising the concern about bursal integrity after vaccination. IBD vaccination using intermediate plus vaccine strains can temporarily deplete the bursal follicles and interrupt the normal B-cell development; if the damage is reversible this process can be followed by B-cell repopulation and histological regeneration. In order to assess this bursal restoration process, specific pathogen free birds were vaccinated with intermediate and intermediate plus IBDV vaccine and bursas were evaluated by histopathology and immunohistochemistry. Both B and T cells were detected in the recovering bursas. At the end of the trial, signs of bursal regeneration and B cell repopulation were observed in the intermediate IBDV vaccinated birds. The bursal restoration process was impaired or delayed in the intermediate plus vaccine group. Relevance of B and T cell repopulation is discussed.

La enfermedad infecciosa de la bolsa (por sus siglas en Inglés IBD) es una enfermedad viral aguda y contagiosa que afecta a los pollos jóvenes, caracterizada por diarrea, picado de la cloaca, temblores, incoordinación, inflamación seguida de atrofia de la bolsa de Fabricious y por grados variables de inmunosupresión. La enfermedad es causada por el virus de la enfermedad infecciosa de la bolsa (por sus siglas en Inglés IBDV) que basado en sus características antigénicas y de patogenicidad ha sido clasificado en cepas clásicas (virus suaves, intermedios e intermedios plus), IBDV muy virulento y cepas variantes. Debido a la amplia presencia de IBDV muy virulento, la industria avícola ha implementado la utilización de vacunas menos atenuadas, lo que genera preocupación por la integridad de la bolsa posterior a la vacunación. La vacunación contra IBD utilizando vacunas intermedias plus puede despoblar los folículos de la bolsa e interrumpir el desarrollo normal de las células B, si el daño es reversible este proceso puede ser seguido de la repoblación de la bolsa con células B y de regeneración histológica. Con la finalidad de evaluar este proceso de restauración, se vacunaron aves libres de patógenos específicos con vacunas intermedia e intermedia plus contra IBDV y se evaluaron las bolsas mediante histopatología e inmunohistoquímica. En las bolsas en recuperación se detectaron tanto células B como células T. Al final del experimento, en las aves vacunadas con la cepa intermedia se observaron signos de regeneración de la bolsa y repoblación de células B. El proceso de restauración de la bolsa se vio comprometido o retrasado en el grupo vacunado con la cepa intermedia plus. Se discute la relevancia de la repoblación de la bolsa con células T y B.

Protection conferred by coarse spray vaccination against challenge with infectious bursal disease virus in commercial broilers.

The efficacy of coarse spray vaccination against pathogenic infectious bursal disease virus (IBDV) in commercial broilers was evaluated. Different coarse spray vaccination schedules using a commercial 2512 strain vaccine were compared with single or double drinking water application at 1 and/or 10 days of age. At 29 days of age, the chickens were challenged with the virulent Edgar strain of IBDV. Seven days postchallenge, severe gross bursal atrophy was observed in the unvaccinated-challenged birds. After challenge and regardless of the method of vaccination used, moderate-to-severe lymphoid depletion was observed, indicating challenge virus replication, later confirmed by reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism analysis. Coarse spray and drinking water vaccination induced protection against body weight loss. Significant differences (P < 0.05) were observed between the unvaccinated-challenged group (1483 g) and the birds vaccinated at 10 days of age by coarse spray (1812 g). The coarse spray vaccination also induced protection against challenge-induced gross bursal atrophy, as determined by bursal index values. After challenge, significant bursal atrophy was observed in the birds orally vaccinated at 1 day (0.61), 10 days (0.66), and 1 and 10 days (0.63) as well as the unvaccinated-challenged birds (0.62), but not in the coarse-spray-vaccinated groups that exhibited bursal indexes above 0.70 and did not differ from the unvaccinated-unchallenged control group. These results suggest that coarse spray vaccination can be considered as another tool to control IBDV in the field.

The VG/GA strain of Newcastle disease virus: mucosal immunity, protection against lethal challenge and molecular analysis.

The Villegas-Glisson/University of Georgia (VG/GA) strain of Newcastle disease virus (NDV) isolated from the intestine of healthy turkeys has been proposed to replicate in the respiratory and intestinal tract of chickens. In the present study, the virus distribution, the mucosal and systemic immune response, the efficacy against lethal challenge and the full genome sequence of the VG/GA strain were compared with the La Sota strain of NDV. The VG/GA strain was detected at different time points in the respiratory and intestinal tract of chickens with a preferential tropism for the latter. Both the VG/GA and La Sota strains induced NDV-specific immunoglobulin A (IgA) at the upper respiratory tract. IgA levels were higher in the trachea for the La Sota strain, while they were higher in the bile and intestine for the VG/GA strain. Positive correlation between virus distribution of the viruses and IgA production was observed. Despite the presence of the maternal antibodies in broilers, early vaccination with the VG/GA strain afforded 95% to 100% protection against lethal challenge, equivalent to the protection conferred by the La Sota strain. Full genome sequence analysis classified the VG/GA strain within class II, genotype II viruses, which also include most of the respirotropic vaccine strains. Differences with the La Sota strain at the nucleotide and amino acid levels that may explain the differential phenotype of the VG/GA were observed; however, verification of the significance of those changes is required. Taken together, these results validate field observations on the efficacy of VG/GA vaccination and demonstrated the unique characteristics of the strain.

Genomic comparison of the complete coding and intergenic regions of the VG/GA Newcastle disease virus and its respirotropic clone 5.

The complete genome of the Villegas-Glisson/University of Georgia (VG/GA) strain of Newcastle disease virus (NDV) and that of a plaque purified clone (clone 5) exhibiting a respiratory phenotype were sequenced and analyzed. The VG/GA strain, isolated from the intestine of healthy turkeys, replicates in the respiratory and intestinal tract of chickens. It is used worldwide as a vaccine strain and its tissue tropism is extremely important for protection against velogenic viscerotropic NDV which targets both intestinal and respiratory epithelia, inducing severe gross and microscopic damage. The clone 5, a plaque purified clone from the VG/GA strain, cannot be recovered from the intestine of infected birds, suggesting a respirotropic nature. A modified primer sequence-independent amplification method was used to sequence the complete coding regions of both viruses and to assess phylogenetic relationships and genomic basis for phenotype differences. The phylogenetic analysis grouped the VG/GA strain and the clone 5 within class II, genotype II viruses and showed that they are greater than 99.9% identical with only 5 nucleotides differences. Both are closely related to classic vaccine strains, such as LaSota and B1. Only 3 amino acid differences at the fusion protein differentiated the VG/GA strain from the clone 5. These differences may explain the differential phenotype observed in the VG/GA strain and are discussed.

A recombinant avian adeno-associated virus as a vector factor for infectious bursal disease vaccination

El virus de la enfermedad infecciosa de la bolsa es una enfermedad de distribución mundial que afecta a aves jóvenes que debe ser controlada mediante vacunación y constituye una de las preocupaciones principales de la industria avícola mundial. Los virus adeno-asociados aviares son virus no patogénicos capaces de dar cabida a porciones relativamente largas de ADN y de infectar una amplia variedad de tipos celulares. Un miembro de esta familia, el virus adeno-asociado aviar ha sido caracterizado por completo y utilizado como un vector para entrega de genes en células y tejidos de embriones de pollo. En el presente estudio se demostró mediante inmunohistoquímica y microscopia electrónica la factibilidad de generar virus adeno-asociados recombinantes expresando la proteína viral 2 del virus de la enfermedad infecciosa de la bolsa. Luego de la inoculación in ovo o intramuscular de aves libres de patógenos específicos con el virus recombinante, se observó evidencia serológica de la expresión de la proteína VP2. La utilización de virus adeno-asociado aviar recombinantes para la entrega de genes es una opción interesante para la vacunación de aves domésticas.

Infectious bursal disease is a worldwide distributed immunosuppressive disease of young chickens that need to be controlled by vaccination; it represents one of the main concerns for the poultry industry. The adeno-associated viruses are non-pathogenic viruses, capable of accommodating relatively long pieces of DNA, and of infecting a wide variety of cell types. A member of this family, the avian adeno-associated virus has been fully characterized and successfully used for gene delivery in chicken embryo tissues and cells. In this study, it was demonstrated by electron microscopy and immunocitochemistry the feasibility of generating recombinant avian adeno-associated virus (rAAAV) virions expressing the immunogenic viral protein 2 of infectious bursal disease virus (IBDV). Serological evidence of VP2 protein expression measured as IBDV specific antibody response after in ovo or intramuscular inoculation of the recombinant virus in specific pathogen free (SPF) chickens was observed. The use of rAAAV virions for gene delivery in poultry is a promising approach to poultry vaccination.

Evaluation of FTA paper and phenol for storage, extraction and molecular characterization of infectious bursal disease virus.

Infectious bursal disease virus (IBDV) is an important poultry pathogen and is distributed world wide that can cause immune suppression and lesions of the bursa of Fabricius. The main component of the virus, VP2, is not only responsible for the bird's immune response, but is important for the molecular identification of this virus as well. The nucleic acid of the virus must be adequately preserved to be analyzed by reverse-transcriptase PCR (RT-PCR) and sequenced for the molecular characterization of the field strain. Phenol inactivation has been the standard for IBDV tissue collection and international shipment; however, there have been some reports of interference with molecular detection capabilities when using phenol. Phenol is also a hazardous chemical and must be handled and shipped carefully. The ability to use the Flinders Technology Associates filter paper (FTA card) for inactivation of several avian pathogens has been proven previously, however no work has been published on its use in IBDV nucleic acid detection. Bursas from experimentally infected birds was imprinted on FTA cards, and then placed in phenol. Samples were evaluated and compared based on molecular detection capabilities between the two inactivation methods. The nucleic acid of the virus was detected in 85% of the FTA card inactivated samples compared to 71% in the phenol inactivated samples. Sequence analysis was performed on samples inactivated by both methods and no differences were found. When comparing the RNA stability at different temperatures, euthanized IBDV infected birds were held at two different temperatures before sampling. No differences were detected for FTA sampling; however, for tissues in phenol the nucleic acid was only detectable up to 2 h post-mortem in the tissues held at 4 degrees C prior to sampling. These findings indicate that the FTA card is an efficient and reliable alternative collection method for molecular detection and characterization of IBDV.

Use of FTA filter paper for the molecular detection of Newcastle disease virus.

The feasibility of using Flinders Technology Associates filter papers (FTA cards) to collect allantoic fluid and chicken tissue samples for Newcastle disease virus (NDV) molecular detection was evaluated. Trizol RNA extraction and one-step reverse transcriptase-polymerase chain reaction (RT-PCR) were used. FTA cards allowed NDV identification from allantoic fluid with a titre of 10(5.8) median embryo lethal doses/ml. The inactivated virus remained stable on the cards for 15 days. NDV was detected from FTA imprints of the trachea, lung, caecal tonsil and cloacal faeces of experimentally infected birds. RT-PCR detection from FTA cards was confirmed by homologous frozen-tissue RT-PCR and virus isolation. Direct nucleotide sequence of the amplified F gene allowed prediction of NDV virulence. No virus isolation was possible from the FTA inactivated samples, indicating viral inactivation upon contact. The FTA cards are suitable for collecting and transporting NDV-positive samples, providing a reliable source of RNA for molecular characterization and a hazard-free sample.

Utilización de las tarjetas FTA® para el diagnóstico molecular del virus de la enfermedad de NEWCASTLE en muestras de fluido alantoideo / Utilization of FTA® Cards for the Molecular Diagnostic of Newcastle Disease Virus in Allantoic Fluid Samples

Se evaluó la factibilidad de utilizar las tarjetas FTA® (Flinders Technology Associates) para la inactivación y transporte de fluido alantoideo infectado con el virus de la enfermedad de Newcastle (VEN). La tarjetas FTA® fueron impregnadas con diluciones seriadas de fluido alantoideo con un título inicial de 10(8,8) DL/50 del VEN cepa LaSota y analizadas mediante la prueba de reacción en cadena por la polimerasa transcriptasa reversa (por sus siglas en ingles RT-PCR) a las 24 horas, 7 y 14 días. La concentración más baja del virus detectada en el fluido alantoideo fue 10(5,8) DL/50. La detección del virus a partir de la tarjeta fue posible hasta 14 días después de su inactivación. El re-aislamiento viral en huevos embrionados a partir de las tarjetas resultó negativo. La inactivación del virus en las tarjetas no afectó la calidad de la secuencia de nucleótidos, permitiendo la determinación de su virulencia mediante la secuenciación de los nucleótidos que codifican la zona de corte de la proteína de fusión resultando ser lentogénico en concordancia con la cepa inicial de virus aplicada a las tarjetas. Se concluye que las tarjetas FTA® representan una alternativa válida para el muestreo, inactivación y diagnóstico molecular del VEN, con un alto grado de bioseguridad.

The feasibility of using FTA® cards (Flinders Technology Associates) for inactivation and transportation of allantoic fluid infected with Newcastle disease virus (NDV) was evaluated. Serial dilutions of allantoic fluid with a titer of 10(8.8) ELD/50 of LaSota strain NDV were loaded on the FTA® cards and analyzed after 24 hour, 7 and 14 days using reverse transcriptase-polimerase chain reaction (RT-PCR). The lowest virus concentration that could be detected from the FTA® cards was 10(5.8) ELD/50. The detection of the inactivated virus was possible after 14 days of virus inactivation. No virus re-isolation in embryonating eggs was possible from the cards. No negative effects of the FTA® card inactivation on the nucleotide sequences were observed, allowing the determination of its virulence by direct nucleotide sequencing of the F protein cleavage site, resulting in a lentogénic strain in concordance with the initial virus applied on the cards. It can be conclude that FTA® cards are a valid alternative for NDV sampling, inactivation and molecular diagnostic with a high degree of biosecurity.

Evaluación de dos planes de vacunación contra la enfermedad de newcastle en pollos de engorde de la línea ross criados bajo condiciones de campo en el estado Zulia Venezuela / Evaluation of two vaccination programs against newcastle disease in ross line broiler chickens reared under field conditions in Zulia state Venezuela

Bajo condiciones de campo del estado Zulia, se probó el efecto de dos planes de vacunación contra la enfermedad de Newcastle (ENC). Se usaron tres grupos de 200 pollos Ross de un día de edad. el primer día se evaluaron serológica e histopatológicamente 20 pollos provenientes del lote a ser utilizado para determinar la calidad inicial y los anticuerpos maternales (AM) contra ENC. Los tratamiento aplicados fueron: T1 Control sin vacunación contra ENC; T2 Hitchner B1B1 por aspersión; T3 cepa enterotrópica (VG/GA) por aspersión + Oleosa subcutánea al día 1, ambos con refuerzos al día 7 y 14 con cepa La Sota en spray y agua respectivamente. En 16 aves de cada tratamiento se estudiaron semanalmente los anticuerpos posvacunales contra ENC a través de Inhibición de la Hemoaglutinación (HI), la respuesta al desafío (día 37) con una cepa velogénica viscerotrópica de ENC (RD) y el grado de alteracción histopatológica en las aves (GL). La data se analizó a través de ANOVA y LS means del paquete estadístico SAS. Los AM promediaron una media geométrica del título (MGT) de 120, no se observaron lesiones hitopatológicas previas al ensayo. Para el T1, T2 y T3 los resultados promediaron una MGT de 7,5/10,7/13,5. En la variable GL no hubo diferencias entre tratamientos, sin embargo se observaron lesiones compatibles con Marek, Gumboro y Micotoxicosis; la RD fue 0 por ciento, 60 por ciento y 90 por ciento de protección respectivamente. La respeuesta serológica a las primeras vacunciones fue inhibida probablemente debido a los altos AM, mientras que la respuesta inmune primaria fue inducida por la tercera vacunación y afectadas por las condiciones de campo. La respuesta inmune humoral y la resistencia al desafío fueron mayores en el T3. Se concluye que bajo condiciones de campo se debe utilizar el plan de vacunación correpondiente al T3