RESUMEN
Careful selection and observance of standard field and laboratory protocols are critical for successful detection and characterization of avian influenza viruses (AIV) from wild birds. Cloacal swabs were collected from hunter-killed or nesting waterfowl and shorebirds from wildlife refuges in Alabama, Georgia, and Florida during 2006 to 2008. Swab samples were inoculated into embryonated eggs followed by hemagglutination (HA) test to determine the presence of hemagglutinating agents. Antigen capture-ELISA (AC-ELISA) and real-time reverse transcription-PCR (RRT-PCR) were used to detect AIV from both allantoic fluids (AF) and swab specimens of HA-positive samples. Hemagglutination inhibition test was used to detect Newcastle disease virus, another hemagglutinating virus common in wild birds. The HA-positive AF were sent to the National Veterinary Services Laboratory for subtyping of the isolates. Out of 825 samples tested, 19 AIV and 3 avian paramyxovirus subtypes were identified by the National Veterinary Services Laboratory. Without egg passage, AC-ELISA did not detect virus, whereas matrix gene of 13 AIV were detected using RRT-PCR. When testing was done on AF, 14 were positive for influenza A by AC-ELISA and 20 by RRT-PCR. Antigen capture-ELISA did not detect influenza A when the HA titer was lower than 125, whereas RRT-PCR detected AIV from AF with HA titer as low as 4. The highest isolation rate was from Florida, where out of 109 samples analyzed, 14 AIV were detected by RRT-PCR from AF. Real-time reverse transcription-PCR was more sensitive, specific, and cost-effective than AC-ELISA. However, to avoid false-negative results, testing should be performed on AF and not directly from cloacal swabs. Our procedures to detect AIV directly from cloacal swabs need further optimization for improved sensitivity.
Asunto(s)
Anseriformes , Virus de la Influenza A/clasificación , Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/virología , Animales , Animales Salvajes , Cloaca/virología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Pruebas de Hemaglutinación/veterinaria , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/veterinariaRESUMEN
Cloacal swabs were taken from migratory hunter-killed, nonmigratory, nesting waterfowl and migratory shorebirds from wildlife refuges in Alabama, Georgia, and Florida during 2006 to 2008. Samples were processed in embryonated eggs followed by hemagglutination (HA), Directigen, and real-time reverse transcription-PCR tests. Sequence analysis of the hemagglutinin (H) gene of the H10N7 Alabama isolate revealed that it was closely related (98%) to recent isolates from Delaware and Canada, but only 90% related to an H10N7 isolated 30 yr ago. Four isolates had 94 to 97% similarity to published H1N1 isolates including one from swine. No H5 or H7 isolates were found. One sample was highly pathogenic in embryos, produced a high HA titer, and was positive for both avian influenza (AIV) and Newcastle disease virus or avian paramyxovirus (APMV)-1. In recent (2008) sampling, more (14%) AIV, APMV, or both were isolated than in 2006 to 2007 (1% isolation rate). The higher isolation rate during 2008 may be attributed to optimized sample collection, storage in dry ice, new egg incubator, healthier eggs, time or habitat for isolation, species sampled, migratory status of birds, and more experience with detection procedures. An additional egg passage resulted in increased viral titer; however, no HA-negative samples became HA positive. The chance of transmission of APMV or low-nonpathogenic AIV from wild waterfowl to commercial poultry is possible. However, the chance of transmission of H5 or H7 AIV isolates from waterfowl to commercial farms in Alabama, Georgia, or Florida is unlikely. Therefore, continual testing of these birds is justified to ensure that H5 or H7 AIV are not transmitted to commercial poultry.
Asunto(s)
Anseriformes/virología , Gripe Aviar/epidemiología , Infecciones por Paramyxoviridae/veterinaria , Paramyxoviridae/aislamiento & purificación , Animales , Animales Salvajes , Paramyxoviridae/genética , Infecciones por Paramyxoviridae/epidemiología , Filogenia , Sudeste de Estados Unidos/epidemiologíaRESUMEN
Two infectious bursal disease viruses (IBDVs 1174 and V1) were isolated from IBDV-vaccinated broiler flocks in California and Georgia. These flocks had a history of subclinical immunosuppression. These isolates are commonly used in IBDV progeny challenge studies at Auburn, AL, as well as vaccine manufacturer's vaccine efficacy studies, because they come from populated poultry-producing states, and are requested by poultry veterinarians from those states. Nested polymerase chain reaction (PCR) generated viral genome products for sequencing. A 491-bp segment from the VP2 gene, covering the hypervariable region, from each isolate was analyzed and compared with previously sequenced isolates. Sequence analysis showed that they were more closely related to the Delaware (Del) E antigenic variant than they are to the Animal Health Plant Inspection Service (APHIS) standard, both at the nucleotide level (96%, 97%) and at the amino acid level (94%, 97%). Both isolates had the glutamine to lysine shift in amino acid 249 which has been reported to be critical in binding the virus neutralizing Mab B69. Phenotypic studies showed that both isolates produced rapid atrophy of the bursae and weight loss, without the edematous bursal phase, in 2-wk-old commercial broilers having antibody against IBDV. A progeny challenge study showed both isolates produced more atrophy of the bursae (less percentage of protection) than the Del E isolate. Molecular and phenotypic data of these important IBDV isolates help in the improved detection and control of this continually changing and important viral pathogen of chickens.
Asunto(s)
Infecciones por Birnaviridae/veterinaria , Pollos/virología , Virus de la Enfermedad Infecciosa de la Bolsa/genética , Virus de la Enfermedad Infecciosa de la Bolsa/fisiología , Proteínas Estructurales Virales/genética , Secuencia de Aminoácidos , Animales , Infecciones por Birnaviridae/virología , Virus de la Enfermedad Infecciosa de la Bolsa/clasificación , Datos de Secuencia Molecular , Fenotipo , Filogenia , Organismos Libres de Patógenos Específicos , Proteínas Estructurales Virales/químicaRESUMEN
This article reviews transmissible proventriculitis in poultry from 1971 to 2006. The disease is important in commercial broilers worldwide, resulting in reduced profits. The aetiology of this disease is unknown and different clinical presentations often result in a confused or complicated diagnosis. The lesion of enlarged proventriculus is often referred to as proventriculitis. However, the term proventriculitis can only be used correctly when there is microscopic evidence of inflammation of the proventriculus glands. Infectious and non-infectious causes of proventriculitis, with major emphasis on the infectious or transmissible causes, are reviewed.
Asunto(s)
Pollos , Enfermedades de las Aves de Corral/patología , Enfermedades de las Aves de Corral/transmisión , Proventrículo/patología , AnimalesRESUMEN
Avian reoviruses (ARVs) can result in disease and economic losses in the poultry industry. Vaccines against ARV may not provide full protection and can cause adverse reactions. The coding sequence of the sigma C protein from strain S1133 of avian reovirus was expressed in Schizasaccharomyces pombe. Sigma C protein expression was demonstrated by Western blotting, and the protein was evaluated for its ability to protect specific-pathogen-free (SPF) chickens against challenge with the virulent S1133 strain. Serologic and challenge-infection data showed the efficacy of the recombinant vaccine administered orally each week for 3 consecutive wk. Sigma C protein induced antibody, as determined by enzyme-linked immunosorbent assay. Percentage (%) protection induced by the low dose (125 microg purified yeast-expressed sigma C protein/chicken) or the high dose (250 microg purified yeast-expressed sigma C protein/chicken) was 64 and 91, respectively. The commercial vaccine administered once or twice provided 82% protection. Results supported the feasibility of a plant-derived vaccine for use in poultry immunization schemes.
Asunto(s)
Proteínas Portadoras/metabolismo , Pollos , Orthoreovirus Aviar/inmunología , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/virología , Infecciones por Reoviridae/veterinaria , Vacunas Sintéticas , Animales , Secuencia de Bases , Western Blotting/veterinaria , Proteínas Portadoras/genética , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática/veterinaria , Vectores Genéticos , Datos de Secuencia Molecular , Pruebas de Neutralización/veterinaria , Orthoreovirus Aviar/genética , Infecciones por Reoviridae/inmunología , Schizosaccharomyces/metabolismo , Análisis de Secuencia de ADN/veterinaria , Organismos Libres de Patógenos EspecíficosRESUMEN
VP2 protein is the major host-protective immunogen of infectious bursal disease virus (IBDV) of chickens. Transgenic lines of Arabidopsis thaliana expressing recombinant VP2 were developed. The VP2 gene of an IBDV antigenic variant E strain was isolated, amplified by RT-PCR and introduced into a plant expression vector, pE1857, having a strong promoter for plant expression. A resulting construct with a Bar gene cassette for bialaphos selection in plant (rpE-VP2) was introduced into Agrobacterium tumefaciens by electroporation. Agrobacterium containing the rpE-VP2 construct was used to transform Ar. thaliana and transgenic plants were selected using bialaphos. The presence of VP2 transgene in plants was confirmed by PCR and Southern blot analysis and its expression was confirmed by RT-PCR. Western blot analysis and antigen-capture ELISA assay using monoclonal anti-VP2 were used to determine the expression of VP2 protein in transgenic plants. The level of VP2 protein in the leaf extracts of selected transgenic plants varied from 0.5% to 4.8% of the total soluble protein. Recombinant VP2 protein produced in plants induced antibody response against IBDV in orally-fed chickens.
Asunto(s)
Arabidopsis/genética , Arabidopsis/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Estructurales Virales/biosíntesis , Proteínas Estructurales Virales/genética , Animales , Infecciones por Birnaviridae/tratamiento farmacológico , Infecciones por Birnaviridae/inmunología , Pollos , Regulación de la Expresión Génica de las Plantas/fisiología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéutico , Proteínas Estructurales Virales/inmunología , Proteínas Estructurales Virales/uso terapéutico , Vacunas Virales/biosíntesis , Vacunas Virales/genética , Vacunas Virales/inmunología , Vacunas Virales/uso terapéuticoRESUMEN
We investigated the effect of in ovo administered reovirus vaccines on the immune responses of specific-pathogen-free chickens. T-cell mitogenic responses to concanavalin A were numerically lower at 9 and 12 days of age and significantly lower at 6 days of age in birds vaccinated with a commercial reovirus vaccine compared with unvaccinated birds or birds vaccinated with an experimental reovirus-antibody complex vaccine. There were no significant differences in proportions of subpopulations of helper (CD4+CD8-) or cytotoxic (CD4-CD8+) T cells except at 12 days of age, when the percentages of CD4-CD8+ cells in the two vaccinated groups were statistically higher than in the nonvaccinated group. B-cell populations were not different among vaccine groups except at 9 days of age, when the vaccinated groups had the highest level of B cells. This commercial reovirus vaccine should not be given in ovo to embryos having little or no maternal antibody, otherwise immunosuppression may occur in the chicks. The addition of the antibody complex to the vaccine prevented this T-cell immunosuppression.
Asunto(s)
Pollos/inmunología , Orthoreovirus Aviar/inmunología , Vacunas Virales/administración & dosificación , Animales , Complejo Antígeno-Anticuerpo/administración & dosificación , Antígenos Virales/administración & dosificación , Linfocitos B/inmunología , Embrión de Pollo , Tolerancia Inmunológica , Activación de Linfocitos , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/prevención & control , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/prevención & control , Infecciones por Reoviridae/veterinaria , Organismos Libres de Patógenos Específicos , Subgrupos de Linfocitos T/inmunologíaRESUMEN
Two experiments determined the influence of an experimental reovirus-antibody complex vaccine on Mareks disease virus (MDV) vaccine when used in ovo. Designs were the same except that specific-pathogen-free (SPF) broiler eggs were used in Experiment 1 and commercial broiler eggs with maternal antibodies against reovirus were used in Experiment 2. At 18 days of incubation, embryos were separated into four groups and inoculated with either diluent, MDV vaccine, reovirus-antibody complex vaccine, or a combination of reovirus-antibody complex and MDV vaccine. At 5 days of age, half the chickens in each group were challenged with MDV. At 7 wk old, all were euthanatized, weighed, and examined. At 7 days of age, remaining chickens in each group were challenged with reovirus. At 21 days old, chickens were euthanatized and weighed. No vaccine adversely affected hatchability or posthatch mortality in SPF or commercial chickens. There were no significant differences in protection against reovirus challenge when vaccines were used separately or in combination, and lesion scores were nearly identical in all vaccinated groups in both experiments. However, percentage of protection against reovirus was lower in Experiment 2, indicating an adverse effect of maternal immunity on efficacy of the reovirus vaccine. There were no significant differences in protection against MDV when the vaccines were used separately or combined. Severity of MDV lesions was nearly identical in all vaccinated groups in both experiments. However, the combination of vaccines gave numerically lower protection against MDV than MDV vaccine alone. Use of a larger number of birds, as in field conditions, may result in statistically lower protection for the vaccine combination. Large field trials are needed to determine the potential of the reovirus-antibody complex vaccine.
Asunto(s)
Anticuerpos Antivirales/sangre , Bovinos/inmunología , Mardivirus/inmunología , Enfermedad de Marek/inmunología , Vacunas Virales/administración & dosificación , Animales , Bovinos/microbiología , Pollos , Enfermedad de Marek/patología , Reproducibilidad de los ResultadosRESUMEN
A commercial reovirus vaccine alone or experimental reovirus vaccine plus antibody complex were inoculated into 18-day-old specific pathogen free (SPF) broiler embryos at 0.1 of the recommended chick dose. The following groups were used: group 1A was not vaccinated or challenged; group 1B was not vaccinated, but was challenged with virulent reovirus; group 2 received the vaccine complexed with 1/4 dilution of antiserum; group 3 received the vaccine with 1/8 dilution of antiserum; group 4 received the vaccine with 1/16 dilution of antiserum, and group 5 received vaccine alone. At 1, 3, 6, 9, and 12 days of age, serum was collected and antibody against avian reovirus was analyzed by enzyme-linked immunosorbent assay (ELISA). At the same times, spleens were collected and vaccine virus detected by inoculating chicken embryo fibroblasts (CEFs) and examining for cytopathic effect. At 15 days of age, chickens in groups 2-5 were challenged with reovirus. At 22 days of age, birds were euthanatized and weighed. Efficacy of the vaccines was based on safety, percent protection, and antibody response. In ovo vaccination with the commercial or experimental vaccines did not adversely affect hatchability of SPF chickens. The vaccine complexed with antibody resulted in significantly less posthatch mortality (3.7%) when compared to mortality of chickens that received vaccine alone (17%). Both vaccine virus recovery and antibody response were delayed at least 3 days in birds receiving the experimental vaccines. In evo administration of reovirus antibody complex vaccines provided at least 70% protection. The experimental reovirus-antibody complex vaccines were safe and efficacious when given in ovo to SPF broiler embryos.
Asunto(s)
Embrión de Pollo/inmunología , Enfermedades de las Aves de Corral/virología , Infecciones por Reoviridae/veterinaria , Reoviridae/inmunología , Vacunas Virales/uso terapéutico , Animales , Embrión de Pollo/virología , Pollos , Ensayo de Inmunoadsorción Enzimática , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/prevención & control , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/prevención & control , Seguridad , Vacunas Virales/administración & dosificación , Vacunas Virales/toxicidadRESUMEN
In ovo vaccination against Marek's disease virus and infectious bursal disease virus (IBDV) in commercial broilers in the United States is common. Little information exists as to the safety and efficacy of intermediate IBDV vaccines given in ovo. Experiments were initiated to determine the safety and efficacy of three commercially available live intermediate IBDV vaccines by in ovo route. Commonly used vaccines were given at 18 days of embryonation to specific-pathogen-free (SPF) broiler embryos (first and second study) or to commercial broiler embryos (third study) that had maternal antibody against IBDV. When any of the antigenic standard vaccines was given at full dose to SPF embryos, embryonic and 3-wk posthatch mortality increased. Vaccines also caused significant microscopic lesions in the bursa of Fabricius at 1 and 3 wk posthatch. In contrast, there was no adverse effect on embryonic or posthatch mortality when vaccines were given at half dose to SPF or commercial broiler embryos. However, significant microscopic lesions were evident at 1 and 3 wk posthatch in the bursae of SPF embryos given the vaccines at half dose. When vaccines were given at half dose to commercial broiler embryos, lesions were evident at 1 but not 3 wk of age. In the third study, in ovo vaccinated chickens were challenged with either a virulent standard (APHIS) or antigenic variant (variant E) IBDV virus at 3 wk of age. All vaccines produced at least 87% protection against the standard and 60% protection against the variant challenge IBDV, as measured by bursal weight to body weight ratios. This study was the first to examine the safety and efficacy of the three commonly used intermediate IBDV vaccines given in ovo in protection against standard and antigenic variant IBDV challenge viruses.
Asunto(s)
Infecciones por Birnaviridae/veterinaria , Virus de la Enfermedad Infecciosa de la Bolsa/inmunología , Óvulo/virología , Enfermedades de las Aves de Corral/prevención & control , Vacunación/veterinaria , Vacunas Virales , Animales , Variación Antigénica , Infecciones por Birnaviridae/inmunología , Infecciones por Birnaviridae/prevención & control , Embrión de Pollo , Pollos , Vacunas contra la Enfermedad de Marek , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/virologíaRESUMEN
Control of infectious bursal disease virus (IBDV) by vaccination is important for poultry production worldwide. Two vaccines, an IBDV immune complex (ICX) vaccine and an IBDV-2512 vaccine, were administered at 100 mean embryo infectious dose to specific-pathogen-free 18-day-old broiler embryos in ovo. At 3, 6, 9, 15, and 21 days post in ovo vaccination (PIOV), bursa, spleen, and thymus tissues were collected and analyzed for virus protein by antigen capture chemiluminescent enzyme-linked immunosorbent assay (ELISA). Chicks were bled and antibody titers were determined by the antibody ELISA. At 21 days PIOV, chickens were challenged with a 1:500 dilution of an antigenic standard IBDV strain. At 28 days PIOV, birds were euthanatized and bursa weight:body weight ratios were determined. Embryos vaccinated with either vaccine exhibited 92% hatchability; however, within 1 wk of hatch, birds vaccinated with IBDV-2512 showed 56% mortality, whereas those given IBDV-ICX had only 3.2% mortality. Both IBDV-ICX and IBDV-2512 vaccines were detected in bursa, spleen, and thymus at day 3 PIOV. A 5-day delay in virus replication was observed with IBDV-ICX vaccine. By day 15 PIOV, the IBDV-ICX was no longer detectable in the bursa and spleen but persisted in the thymus. The IBDV-2512 vaccine persisted in the spleen and thymus on day 15 PIOV. By day 21 PIOV, neither vaccine virus was detected in any lymphoid organ. This assay can be useful in the early detection of vaccine virus in the tissues of chickens vaccinated via the in ovo route. Both vaccines caused bursal atrophy at all times PIOV. The IBDV-2512 caused splenomegaly at day 6 PIOV, whereas splenomegaly was not seen in IBDV-ICX-vaccinated birds until day 9 PIOV. Thymus atrophy was observed in IBDV-2512-vaccinated chicks from day 3 PIOV, whereas this occurred on day 15 PIOV in IBDV-ICX-vaccinated birds. Bursa weight: body weight ratios in IBDV-ICX-vaccinated unchallenged and vaccinated challenged birds were not different (P < 0.05).
Asunto(s)
Infecciones por Birnaviridae/veterinaria , Pollos , Virus de la Enfermedad Infecciosa de la Bolsa/aislamiento & purificación , Tejido Linfoide/virología , Enfermedades de las Aves de Corral/prevención & control , Vacunas Virales/administración & dosificación , Animales , Anticuerpos Antivirales/sangre , Complejo Antígeno-Anticuerpo/inmunología , Infecciones por Birnaviridae/inmunología , Infecciones por Birnaviridae/prevención & control , Peso Corporal , Bolsa de Fabricio/virología , Embrión de Pollo , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Virus de la Enfermedad Infecciosa de la Bolsa/inmunología , Mediciones Luminiscentes , Tamaño de los Órganos , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/virología , Organismos Libres de Patógenos Específicos , Bazo/virología , Timo/virología , Replicación ViralRESUMEN
Two monoclonal antibodies (MAb), E9 and H3, prepared against avian reovirus (ARV) S1133, were used in an immuno-dot assay to detect ARV antigens from cell culture and from tendon tissue samples of chickens. The limit of viral antigens detected was 8 ng using both MAb probes. The probes detected 10 ARV isolates representing at least two serotypes or pathotypes. The results indicated that these probes had broad specificity. The probes, however, did not cross-react with viral antigens prepared from six unrelated avian viruses. The ARV antigens in tendon tissue samples were detected by both probes, and it is possible, therefore, to use either of the two MAb probes for detection of ARV infections.
Asunto(s)
Anticuerpos Monoclonales , Antígenos Virales/análisis , Orthoreovirus/aislamiento & purificación , Enfermedades de las Aves de Corral/diagnóstico , Infecciones por Reoviridae/veterinaria , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Antígenos Virales/inmunología , Cápside/inmunología , Células Cultivadas , Pollos , Reacciones Cruzadas , Immunoblotting/métodos , Orthoreovirus/inmunología , Orthoreovirus/fisiología , Infecciones por Reoviridae/diagnóstico , Tendones/virologíaRESUMEN
Development of molecular techniques for the detection of infectious bursal disease virus (IBCV) is an important area of research. An in situ hybridization (ISH) test was developed with a 491-bp cDNA fragment derived from the VP2 gene of IBDV. The fragment was amplified and simultaneously labeled with incorporation of digoxigenin-11-dUTP in a nested polymerase chain reaction (PCR) assay. The resulting digoxigenin-labeled 491-bp nested PCR product was used as probe for ISH to detect and localize IBDV RNA in formalin-fixed, paraffin-embedded bursae of Fabricius from chickens both experimentally infected as well as commercially reared. Bursae from six clinically ill commercial broilers suspected to be IBDV infected were examined by ISH and immunohistochemistry. In two samples, IBDV infection was detected by both ISH and immunohistochemistry, whereas in the other two histologically normal bursae, IBDV was detected only by ISH. Two commercial chickens with atrophied bursae were negative by both ISH and immunohistochemistry. No positive IBDV stained cells were in RNase treated sections from infected birds, uninfected chickens, or reovirus-infected chickens. The ISH test developed herein resulted in important modifications, which makes it superior to other previously published procedures. We also described a direct in situ reverse transcription-polymerase chain reaction method for the amplification and detection of IBDV genome in formalin fixed, paraffin-embedded bursae of Fabricius with a single primer pair with direct incorporation of digoxigenin-11-deoxyuraciltriphosphate (dUTP) into the amplicon. Both molecular tests with their important modifications represent improved detection of IBDV.
Asunto(s)
Infecciones por Birnaviridae/veterinaria , Virus de la Enfermedad Infecciosa de la Bolsa/aislamiento & purificación , Reacción en Cadena de la Polimerasa/veterinaria , Enfermedades de las Aves de Corral/virología , Animales , Infecciones por Birnaviridae/virología , Pollos , Inmunohistoquímica , Hibridación in Situ/veterinaria , Proteínas Estructurales Virales/análisisRESUMEN
A rapid and sensitive protocol for the detection and amplification of infectious bursal disease virus (IBDV) RNA in the bursa of Fabricius was developed. By digestion with proteinase K and subsequent extraction with phenol and chloroform, IBDV RNA was efficiently released from a single bursa. IBDV RNA extraction time was shortened to 4 hr, compared with 24 hr with the traditional method, and only one bursa was needed instead of five. This more simplified procedure resulted in a significant reduction in costs due to less labor and the reduction in expensive chemicals and reagents. Four primers were selected from the sequence of a hypervariable region in VP2 genes. For the amplification of genomic IBDV RNA, the product (643 bp) of reverse transcriptase-polymerase chain reaction (RT-PCR) was reamplified and double checked by a nested PCR amplifying a 491-bp cDNA. The sensitivity of nested PCR was at least 100 times greater than RT-PCR as determined by dilution of the bursal homogenate. The fidelity of the nested PCR product was confirmed by Southern blotting, demonstrating specificity to the VP2 gene of IBDV. The simplified sample processing, shortened procedure time, and technical ease of this nested PCR render it more suitable for implementation in routine RT-PCR with restriction fragment length polymorphism testing for the detection and differentiation of IBDV RNA, especially for studies of IBDV infections of individual chickens.
Asunto(s)
Infecciones por Birnaviridae/veterinaria , Bolsa de Fabricio/virología , Pollos/virología , Virus de la Enfermedad Infecciosa de la Bolsa , Reacción en Cadena de la Polimerasa/métodos , Enfermedades de las Aves de Corral/virología , Animales , Infecciones por Birnaviridae/diagnóstico , Enfermedades de las Aves de Corral/diagnósticoRESUMEN
The VP2 hypervariable region of infectious bursal disease virus (IBDV) from nine Mainland Chinese strains was amplified by reverse transcriptase/nested polymerase chain reaction and cloned into pGEM-T vector. The nine isolates, which were from the center (HN3), the north (Bj-1, B2/28, HD96), the east (JS-18 and AH-2), the northeast (D11-2, C4-2), and the west (Ts) of China, were sequenced and compared with each other and with six reference IBDV sequences. Clustering analysis separated the nine isolate into two groups. The six virulent isolates, propagated in bursae, formed the first group. They revealed only one to three amino acid changes from the very virulent (vv) European and Japanese isolates, suggesting that they might have the same origin as European and Japanese vvIBDV strains. On the basis of their distinct geographic origins, extensive dissemination of vvIBDV in China was indicated. (The other three chicken embryo fibroblast cell cultured isolates with mild pathogenicity were placed in the second group.) Their sequences correlated closely with those of the culture-adapted strains (Cu-1 (4) and Cj-801). None of the nine isolates showed very close sequence relationship with the antigenic variant strains from the USA. Although antigenic variants have been reported in China, the reverse transcriptase/polymerase chain reaction-restriction endonuclease analyses of the nine viruses tested herein were not similar to any U.S.A. variant strains on the basis of computer software analysis. Our results and conclusions agree with a previous molecular study of IBDV isolates from the south of China.
Asunto(s)
Pollos/virología , Variación Genética , Virus de la Enfermedad Infecciosa de la Bolsa/clasificación , Virus de la Enfermedad Infecciosa de la Bolsa/genética , Filogenia , Proteínas Estructurales Virales/genética , Secuencia de Aminoácidos , Animales , Infecciones por Birnaviridae/veterinaria , Infecciones por Birnaviridae/virología , Bolsa de Fabricio/virología , Células Cultivadas , Embrión de Pollo , China , Clonación Molecular , Evolución Molecular , Fibroblastos , Geografía , Virus de la Enfermedad Infecciosa de la Bolsa/aislamiento & purificación , Datos de Secuencia Molecular , Enfermedades de las Aves de Corral/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Proteínas Estructurales Virales/químicaRESUMEN
A nested polymerase chain reaction (PCR) with subsequent nucleotide sequence analysis identified and differentiated avian reoviruses (ARVs). PCR products amplified from the S1 gene segment of ARV of USA isolates were 738 and 342 bp, respectively. PCR products were conformed by Southern and dot blot hybridizations. The amplified cDNA fragments were cloned into the pUC18 vector and subjected to DNA sequencing. The nucleotide and deduced amino acid sequences of four USA (S1133, 1733, 2408, and CO8) and two Australian isolates (RAM-1 and SOM-4) were compared. Results of paired difference analysis and a predicted dendrogram revealed that USA isolates were closely related, but different from, Australian isolates. The deduced amino acid sequences of the N-terminal region of ARV sigma C showed a heptapeptide repeat of hydrophobic residues in all ARV isolates.
Asunto(s)
Orthoreovirus/química , Orthoreovirus/genética , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADN , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Embrión de Pollo , Clonación Molecular , Fibroblastos , Immunoblotting , Datos de Secuencia Molecular , Orthoreovirus/aislamiento & purificación , Filogenia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Proteínas Virales/genéticaRESUMEN
Normal physeal and dyschondroplastic cartilage of broiler chickens was examined for six enzymes by isoelectric focusing in thin-layer polyacrylamide slab gels. Acid phosphatase (ACP), esterase (EST), malate dehydrogenase (MDH), and peroxidase (PRX) were present in the normal physeal cartilage but not in the dyschondroplastic cartilage. Staining intensity of glucose-6-phosphate isomerase (GPI) and triose-phosphate isomerase (TPI) was reduced in the dyschondroplastic cartilage compared with that of the physeal cartilage. Differences in the presence of these enzymes possibly demonstrated their roles in processes of bone formation, cartilage resorption, and calcification. ACP could be involved in calcification. Lack of EST and PRX may be related to the failure of vascular invasion in dyschondroplastic cartilage of afflicted birds. A deficiency of MDH and reduced GPI and TPI in dyschondroplastic cartilage may reflect a reduction in the activity of energetic metabolism, causing the dissipation of energy and necrotic cells.
Asunto(s)
Cartílago Articular/enzimología , Enzimas/análisis , Osteocondrodisplasias/veterinaria , Enfermedades de las Aves de Corral , Fosfatasa Ácida/análisis , Animales , Desarrollo Óseo , Cartílago Articular/fisiología , Cartílago Articular/fisiopatología , Pollos , Esterasas/análisis , Glucosa-6-Fosfato Isomerasa/análisis , Focalización Isoeléctrica , Malato Deshidrogenasa/análisis , Osteocondrodisplasias/enzimología , Osteocondrodisplasias/fisiopatología , Peroxidasas/análisis , Valores de Referencia , Tibia , Triosa-Fosfato Isomerasa/análisisRESUMEN
Avian reoviruses (ARVs) cause important losses in the poultry industry. Improved tests are needed for diagnosis of ARV infections. A cDNA library was prepared from the S1133 isolate of ARV. EcoRI-adaptored cDNA molecules were ligated into the plasmid pUC19 and used to transform Escherichia coli strain DH5 alpha MCR. One cDNA clone was selected and designated HJp1. The HJp1 was labeled by random priming with digoxigenin-dUTP. This cDNA probe hybridized with the S1 gene fragment of the ARV S1133 strain in northern blot hybridization. The probe detected ARV isolates in dot-blot hybridization assays. The probe did not cross-hybridize with nucleic acids extracted from mock-infected chicken embryo fibroblast cells or unrelated avian viruses. Probe HJp1 detected as little as 0.78 ng of ARV RNA.
Asunto(s)
Enfermedades de las Aves de Corral , Infecciones por Reoviridae/veterinaria , Reoviridae/aislamiento & purificación , Animales , Células Cultivadas , Embrión de Pollo , Pollos , Clonación Molecular , Sondas de ADN , ADN Complementario , Genoma Viral , ARN Bicatenario/análisis , ARN Bicatenario/genética , ARN Viral/análisis , ARN Viral/genética , Infecciones por Reoviridae/diagnóstico , Sensibilidad y EspecificidadRESUMEN
An in situ hybridization (ISH) technique using a digoxigenin (DIG)-labeled cDNA probe detected avian reovirus (ARV) RNA in formalin-fixed, paraffin-embedded chicken tissues. Tissues were collected 3 and 10 days following inoculation with the R-2 or the S1133 strain of ARV. The cDNA clone HJp1, located on the S1 gene segment of the ARV S1133 strain, was used to prepare a nonradioactive probe. The ISH assay localized ARV RNA in infected tissues including heart, liver, intestine, pancreas, and tendon. No positive-stained cells occurred in sections from uninfected chickens.
Asunto(s)
Enfermedades de las Aves de Corral , Infecciones por Reoviridae/veterinaria , Reoviridae/aislamiento & purificación , Animales , Pollos , Sondas de ADN , ADN Complementario , Digoxigenina , Formaldehído , Genes Virales , Técnicas Histológicas , Hibridación in Situ , Parafina , ARN Viral/análisis , Infecciones por Reoviridae/patologíaRESUMEN
The VP2 gene is part of the genomic segment A of infectious bursal disease virus (IBDV). It has been identified as the major host-protective antigen of IBDV and is known to contain conformationally dependent protective epitopes. A 643-base pair segment covering the hypervariable region of this gene from three recent serologic variant IBDV isolates from the southeastern United States, two variants from the Delmarva Peninsula, and three serologic standard viruses were amplified and sequenced using the reverse transcription polymerase chain reaction and cycle sequencing techniques. This was done to determine the molecular similarity among isolates that differ antigenically and pathologically. Sequence analysis suggested that the Arkansas (Ark) and Mississippi (Miss) isolates evolved closely and separately from the Delmarva variants (GLS and DELE), in contrast to the other southeastern variant Georgia (Ga), which is more closely related (98.32%) to Delaware E (DELE). All variants, except for Miss, underwent a shift in amino acid number 222 from proline to threonine. The sequence of Univax BD virus, a commercially available intermediate vaccine, was markedly different, evolving from a separate lineage than the others. Restriction enzyme sites could differentiate most isolates. Except for Miss, variants do not have EcoRII site at the larger hydrophilic domain. All variants lost their HaeIII, StuI, and StyI cutting sites with a change in base number 856. The TaqI site is in DELE, whereas the SpeI site is absent in the standard vaccine viruses. The SWASASGS heptapeptide is conserved in all virulent viruses, including APHIS, but not in the attenuated (Univax BD and Bursa Vac 3) and published (D78 and PBG98) vaccines.