Percepción del riesgo en el proceso de producción de vacunas aviares / Risk perception in the avian vaccine production process

La importancia que tienen para la avicultura cubana el virus de la enfermedad infecciosa de la bolsa (Gumboro) y el virus de la viruela aviar, así como la producción de vacunas que permitan controlar las enfermedades producidas por estos agentes biológicos, justifican la necesidad del establecimiento de una buena gestión de la bioseguridad, ya que el desconocimiento de los peligros y riesgos del personal que labora en estas vacunas puede provocar accidentes de consecuencias indeseables para el producto, escapes de estos microorganismos durante sus procesos productivos y la consecuente contaminación del medio ambiente. El objetivo de la presente investigación fue realizar un análisis de la percepción de riesgo existente en el personal responsable del proceso de producción de dos vacunas aviares. Para ello se utilizó el software RISKPERCEP en una instalación de producción de vacunas aviares; su aplicación mostró variables que demostraron subestimación del riesgo por el personal expuesto y variables con tendencia a la sobrestimación, asociadas fundamentalmente al incorrecto diseño de la instalación. Se concluye que existe la necesidad de una buena capacitación y que se impartan cursos de actualización de bioseguridad donde se tengan en cuenta todos los aspectos del diseño del laboratorio que puedan solucionarse(AU)

The importance of infectious bursal disease virus and fowl pox virus for Cuban poultry farming, as well as the production of vaccines to control the diseases caused by these biological agents, justifies the need for establishment of a good Biosafety management; since the ignorance of the dangers and risks on the part of the personnel that works in them can cause accidents with undesirable consequences for the product, escapes of these microorganisms during their production processes and the consequent contamination of the environment. The objective of the research was to carry out an analysis of the perception of risk in the personnel responsible for the production process of two avian vaccines. The RISKPERCEP software was used in an avian vaccine production facility; its application showed variables that demonstrated underestimation of the risk by the exposed personnel and variables with a tendency to overestimate; fundamentally associated with the incorrect design of the facility. Finally, it is proposed that biosafety update courses be given and that all aspects of the laboratory design that can be solved are taken into account(AU)

Immune response elicited by the oral administration of an intermediate strain of IBDV in chickens

The immune response elicited by the oral inoculation of an intermediate strain of infectious bursal disease virus was studied in chickens. A strong over expression of IL-6, IL-8, IFNα and IFNγ was observed in bursa at 3 days post inoculation together with an increase in splenic NO2 release. An influx of T-lymphocytes was also detected.

Efficient assembly of full-length infectious clone of Brazilian IBDV isolate by homologous recombination in yeast

The Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens. Advances in molecular virology and vaccines for IBDV have been achieved by viral reverse genetics (VRG). VRG for IBDV has undergone changes over time, however all strategies used to generate particles of IBDV involves multiple rounds of amplification and need of in vitro ligation and restriction sites. The aim of this research was to build the world's first VRG for IBDV by yeast-based homologous recombination; a more efficient, robust and simple process than cloning by in vitro ligation. The wild type IBDV (Wt-IBDV-Br) was isolated in Brazil and had its genome cloned in pJG-CMV-HDR vector by yeast-based homologous recombination. The clones were transfected into chicken embryo fibroblasts and the recovered virus (IC-IBDV-Br) showed genetic stability and similar phenotype to Wt-IBDV-Br, which were observed by nucleotide sequence, focus size/morphology and replication kinetics, respectively. Thus, IBDV reverse genetics by yeast-based homologous recombination provides tools to IBDV understanding and vaccines/viral vectors development.

Effects of Live Infectious Bursal Disease Vaccines, on Immune Response of Vaccinated Chicks.

Aim: Relationship between virus titers of live Infectious Bursal disease (IBD) vaccines and their serum-conversion abilities was studied. Study design and Methodology: Five batches of each, of five IBD vaccine brands used in Nigeria, were tested for virus titers. Each of the vaccine brands was also used to vaccinate a group of fifteen 12-days old chicks to study their serum-conversion abilities. Mean antibody titers of the groups of chicks were plotted, on a graph, against virus titers of the vaccine brands used to vaccinate them. Results: Mean Modified Passive Haemagglutination titers of IBD virus in the vaccines,were:1,065.60±780.03,1,472.00±748.55,2,112.00±1984.00,2,176.00±1920. 00 and 2,585.00±926.92 while mean antibody titers they elicited were, 1,356.80±241.51, 1,280.00±174.88, 448.00±79.25, 998.40±196.27 and 332.80±51.20, respectively. Line of best fit of graph of antibody titers of vaccinated chicks on vaccine titers, showed that reducing titers of the live IBD vaccines improved their immunogenicity. Conclusions: The inverse relationship between virus titers of the vaccines and their serum conversion abilities, suggests that, if viral titers of live IBD vaccines are too high, immune-suppression instead of enhancement of immune response may occur.

Partial VP1 sequencing of Brazilian infectious bursal disease virus strains

Infectious bursal disease virus (IBDV) is classified according to the antigenicity and virulence into classical virulent (cv), very virulent (vv), and antigenic variant strains. The molecular basis for the IBDV antigenic variation is well established and is associated to the capsid protein, VP2 (gene VP2 of segment A), whereas both VP2 and the RNA-dependent RNA polymerase, VP1 (gene VP1 of segment B), have been correlated with the virulence. In this study, seventeen Brazilian IBDV samples previously characterized by the VP2 gene as cv (three) and vv (fourteen) strains were genetically and molecularly analyzed for their VP1 gene. All of the strains kept with the same cv or vv classification except one sample, Br/03/DR. This sample was classified as vv by its VP2 gene, but it was most closely related to the cv strains by its VP1 partial sequence and phylogeny. Studies on the phylogeny of VP1 have suggested a possible reassortment event that originated the vvVP1. In this case, the sample carrying vvVP2 and cvVP1 could be a descendant of IBDV ancestors prior to the reassortment of vvVP1; alternatively, it could be the result of a genetic exchange between the segments of different strains or with a live attenuated vaccine. Nevertheless, this is the first report of natural genetic reassortment of IBDV in Brazil.

Investigation on Newcastle Disease Virus (NDV), Infectious Bursal Disease Virus (IBDV) and Avian Poxvirus (APV) in magellanic penguins in Southern region of Brazil

To investigate the exposure of the Newcastle disease virus (NDV), infectious bursal disease virus (IBDV) and avian poxvirus (APV) in Magellanic penguins found on the beaches in Southern regions of Brazil, the frequency of serum antibodies was estimated in 89 samples taken during 2005 and 2006. All the penguins were negative for the presence of antibodies against NDV by hemagglutination inhibition test and to APV by indirect ELISA. The reactivity was similar to the positives controls using ELISA kit for the IBDV made in the chickens in 50 samples. This reactivity also was demonstrated in 42 samples using agar gel immunodiffusion. No clinical signs related to IBDV infection were observed. The results indicated the absence of infection by NDV and APV but suggested IBDV exposure in the population of penguins studied.

Molecular characterization of two Bangladeshi infectious bursal disease virus isolates using the hypervariable sequence of VP2 as a genetic marker

Two Bangladeshi infectious bursal disease virus (IBDV) isolates collected in 2007, termed GB1 and GB3, were subjected to comparative sequencing and phylogenetic analyses. Sequence analysis of a 474-bp hypervariable region in the VP2 gene revealed that among four major amino acid substitutions observed in the strains, two were unique to GB1 and GB3 (Ser217Leu and Ala270Thr) while one substitution was only found in GB1 (Asn299Ser). Among IBDVs from Bangladesh including GB1 and GB3, the rate of identity and homology was around 97~99%. The amino acid sequences of GB1 and GB3 differ from those of previous Bangladeshi IBDV isolates and contain amino acid substitutions Pro222Ala and Asn299Ser (in GB3 only). Phylogenetic analysis revealed that GB1 and GB3 are grouped with other very virulent IBDVs of European and American origin in contrast to two previously isolated Bangladeshi IBDV strains (GenBank accession Nos. AF362776 and AF260317), which belong to the Asian group. It was concluded that GB1 and GB3 belong to a very virulent group of IBDVs. However, amino acid sequences of GB1 and GB3 differ from those of the other Bangladeshi IBDVs by one or two amino acids encoded in the hypervariable region of the VP2 gene.

Detection of chicken anemia virus and infectious bursal disease virus co-infection in broilers / Detecção do virus da anemia das galinhas em coinfecção com o vírus doença infecciosa bursal em frangos

This survey aimed to investigate chicken anemia virus (CAV) in broilers flocks experimenting retarded growth and increasing mortality since the fourth day of age. Clinically, chickens presented depression, paleness, depigmentation and retarded growth. At necropsy, chickens presented CAV-compatible lesions. Samples from liver, spleen and thymus were tested by PCR for a 675-bp fragment of the CAV VP-1 gene, and all tested samples were positive. Serological and molecular techniques did not detect other pathogens, such as adenovirus, reovirus, astrovirus, infectious bursal disease and avian infectious bronchitis virus. These results showed that chicken anemia virus (CAV) may occur since the first few days of life in broilers - a fact not as yet reported -, associated with high pathogenic Infectious Bursal Disease Virus (IBDV) vaccine strain may induce a persistent growth retarded for several weeks in broilers.

Este estudo investigou a manifestação do vírus da Anemia Infecciosa das Aves (VAIA) em lotes de frangos que apresentavam retardo no crescimento e aumento da mortalidade observado a partir do quarto dia de idade. Clinicamente, as aves apresentavam depresão, palidez, despigmentação e retardo de crescimento. À necropsia, as aves apresentavam lesões compatíveis com a infecção pelo vírus da Anemia infecciosa das aves (VAIA). Amostras de fígado, baço e timo foram examinadas por PCR que amplifica um frangmento de 675 pb do gene VP-1 do VAIA. Todos os órgãos examinados foram positivos para o vírus da Anemia Infecciosa das Aves. Os demais patógenos, como adenovírus, reovírus, astrovírus, vírus da doença infecciosa bursal e coronavírus aviário não foram detectados pelas diferentes técnicas laboratoriais, como sorologia, PCR ou PAGE. Os resultados mostraram que o vírus da Anemia Infecciosa das Aves (VAIA) pode manifestar-se clinicamente nos primeiros dias de vida dos frangos – um fato ainda não reportado – associado ao vírus vacinal da doença infecciosa bursal (DIB) cepa forte pode induzir um persistente retardo de crescimento, por várias semanas, em frangos.

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.

Efficacy of VP2 protein expressed in E. coli for protection against highly virulent infectious bursal disease virus

The ability of a heat-inactivated whole virus from a highly virulent infectious bursal disease virus (hvIBDV) and VP2 protein from hvIBDV expressed in E. coli provided protection against a hvIBDV challenge in specificpathogen- free (SPF) chickens. Six out of seven chickens that were injected three times with crude VP2 protein developed significant antibody titer against IBDV. However, only four out of the seven chickens survived the hvIBDV challenge. Despite showing low antibody titer profiles, all chickens immunized with the heat-inactivated whole virus also survived the challenged with hvIBDV. However, all of these chickens had bursal atrophy and mild to moderate depletion of lymphocytes. Thus, antibodies raised against IBDV VP2 protein expressed in E. coli and denatured IBDV proteins induced some degree of protection against mortality but not against bursal damage following challenge with hvIBDV.

Effects of DDA, CpG-ODN, and plasmid-encoded chicken IFN-gamma on protective immunity by a DNA vaccine against IBDV in chickens

This study examined the adjuvant effects of dimethyl dioctadecyl ammonium bromide (DDA), CpG oligodeoxynucleotides (CpG-ODN), and chicken interferon-gamma (ChIFN-gamma) on a DNA vaccine (pcDNA-VP243) against the infectious bursal disease virus (IBDV). A plasmid encoding chicken IFN-atilde was constructed. Twice at 2-week intervals, twoweek-old chickens were injected intramuscularly and intraperitoneally with either a DNA vaccine alone or a DNA vaccine together with the respective adjuvants. On week 2 after the second immunization, the chickens were orally challenged with the highly virulent IBDV. The groups that received the DNA vaccines plus either DDA or CpG-ODN showed significantly lower survival rates than the group that received the DNA vaccine alone. However, the survival rates for the DNA vaccine alone and for the DNA vaccine plus ChIFN-gamma were similar. The chickens had no detectable antibodies to the IBDV before the challenge but all the surviving chickens in all groups except for the normal control group showed the induction of antibodies to the IBDV at day 10 after the challenge. As judged by the lymphocyte proliferation assays using the a WST-8 solution performed on the peripheral blood and splenic lymphocytes, the stimulation indices (SI) of the peripheral blood lymphocytes in all groups except for the normal control group were similar immediately before the challenge. At 10 days post-challenge, the SI for DNA vaccine plus either CpG-ODN or ChIFN-gamma was similar to that of the DNA vaccine control group. For splenic lymphocytes, the SI in the DNA vaccine plus CpG-ODN and DNA vaccine plus ChIFN-gamma groups were higher than for the DNA vaccine control. These results suggest that DDA actually compromises the protection against the IBDV by DNA vaccine, and CpG-ODN and IFN-gamma had no significant effect.