Detection of avian reovirus (ARV) by ELISA based on recombinant σB, σC and σNS full-length proteins and protein fragments.

Introduction. Avian reovirus (ARV) is associated with arthritis/tenosynovitis and malabsorption syndrome in chickens. The σC and σB proteins, both exposed to the virus capsid, are highly immunogenic and could form the basis for diagnostic devices designed to assess the immunological status of the flock.Gap Statement. Commercial ARV ELISAs cannot distinguish between vaccinated and infected animals and might not detect circulating ARV strains.Aim. We aimed to develop a customized test to detect the circulating field ARV strains as well as distinguish between vaccinated and unvaccinated animals.Methodology. We developed ELISA assays based on recombinant (r) σB, σC and the nonstructural protein σNS and tested them using antisera of vaccinated and unvaccinated chickens as well as negative controls. Fragments of σB and σC proteins were also used to study regions that could be further exploited in diagnostic tests.Results. Vaccinated and unvaccinated birds were positive by commercial ELISA, with no difference in optical density values. In contrast, samples of unvaccinated animals showed lower absorbance in the rσB and rσC ELISA tests and higher absorbance in the rσNS ELISA test than the vaccinated animals. Negative control samples were negative in all tests. Fragmentation of σB and σC proteins showed that some regions can differentiate between vaccinated and unvaccinated animals. For example, σB amino acids 128-179 (σB-F4) and σC amino acids 121-165 (σC-F4) exhibited 85 and 95% positivity among samples of vaccinated animals but only 5% and zero positivity among samples of unvaccinated animals, respectively.Conclusion. These data suggest that unvaccinated birds might have been exposed to field strains of ARV. The reduction in absorbance in the recombinant tests possibly reflects an increased specificity of our test since unvaccinated samples showed less cross-reactivity with the vaccine proteins immobilized on ELISAs. The discrepant results obtained with the protein fragment tests between vaccinated and unvaccinated animals are discussed in light of the diversity between ARV strains.

Avian Reoviruses in Poultry Farms from Brazil.

Avian reovirus (ARV) is highly disseminated in commercial Brazilian poultry farms, causing arthritis/tenosynovitis, runting-stunting syndrome, and malabsorption syndrome in different meat- and egg-type birds (breeders, broilers, grillers, and layers). In Brazil, ARV infection was first described in broilers in the 1970s but was not considered an important poultry health problem for decades. A more concerning outcome of field infections has been observed in recent years, including condemnations at slaughterhouses because of the unsightly appearance of chicken body parts, mainly the legs. Analyses of the performance of poultry flocks have further evidenced economic losses to farms. Genetic and antigenic characterization of ARV field strains from Brazil demonstrated a high diversity of lineages circulating in the entire country, including four of the five main phylogenetic groups previously described (I, II, III, and V). It is still unclear if all of them are associated with different diseases affecting flocks' performance in Brazilian poultry. ARV infections have been controlled in Brazilian poultry farms by immunization of breeders and young chicks with classical commercial live vaccine strains (S1133, 1733, 2408, and 2177) used elsewhere in the Western Hemisphere. However, genetic and antigenic variations of the field isolates have prevented adequate protection against associated diseases, so killed autogenous vaccines are being produced from isolates obtained on specific farms. In conclusion, ARV field variants are continuously challenging poultry farming in Brazil. Epidemiological surveillance combined with molecular biological analyses from the field samples, as well as the development of vaccine strains directed toward the ARV circulating variants, are necessary to control this economically important poultry pathogen.

Reovirus aviares en granjas avícolas de Brasil. El reovirus aviar (ARV) está muy diseminado en las granjas avícolas comerciales brasileñas y causa artritis/tenosinovitis viral, síndrome de retraso de enanismo infeccioso y síndrome de malabsorción en diferentes tipos de aves de carne y huevos (reproductoras, pollos de engorde, parrillas y ponedoras). En Brasil, la infección por reovirus aviares se describió por primera vez en pollos de engorde en la década de 1970, pero no se consideró un problema importante de salud avícola durante décadas. En los últimos años se ha observado un resultado más preocupante de las infecciones de campo, incluidos los decomisos en las plantas de procesamiento debido a la apariencia antiestética de las partes del cuerpo de los pollos, principalmente las patas. Los análisis del desempeño de las parvadas avícolas han evidenciado pérdidas económicas adicionales para las granjas. La caracterización genética y antigénica de las cepas de campo de reovirus aviares de Brasil demostró una gran diversidad de linajes que circulan en todo el país, incluidos cuatro de los cinco grupos filogenéticos principales descritos anteriormente (I, II, III y V). Todavía no está claro si todos ellos están asociados con diferentes enfermedades que afectan el rendimiento de las parvadas en las aves de corral brasileñas. Las infecciones por reovirus aviares se han controlado en granjas avícolas brasileñas mediante la inmunización de reproductores y pollitos jóvenes con cepas vacunales vivas comerciales clásicas (S1133, 1733, 2408 y 2177) utilizadas en otras partes del hemisferio occidental. Sin embargo, las variaciones genéticas y antigénicas de los aislamientos de campo han impedido una protección adecuada contra enfermedades asociadas, por lo que se están produciendo vacunas autógenas inactivadas a partir de aislamientos obtenidos en granjas específicas. En conclusión, las variantes de campo de ARV son un desafío continuo para la avicultura en Brasil. La vigilancia epidemiológica combinada con análisis de biología molecular de las muestras de campo, así como el desarrollo de cepas de vacunas dirigidas a las variantes circulantes de los reovirus aviares, son necesarias para controlar este patógeno avícola económicamente importante.

Emerging new avian reovirus variants from cases of enteric disorders and arthritis/tenosynovitis in Brazilian poultry flocks.

1. The objective of this study was to characterise circulating Brazilian avian reovirus (ARV) strains by genetic analysis of the σC protein encoded by segment 1 of the viral genome and compare these with those of viral strains used for immunising commercial poultry.2. The analysis detected the presence of ARV genomes by quantitative reverse transcriptase PCR (RT-qPCR) in the enteric samples and the joint tissues (JT) of birds with signs of viral arthritis/tenosynovitis. Nucleotide sequencing used 16 strains (three commercial vaccines, 10 from enteric tissues and three from JT). The results indicated high variability in the amino acid sequences of 13 wild strains, showing between 40% and 75% similarity compared with the vaccine strains (S1133 and 2177).3. The sequences were grouped into three well-defined clusters in a phylogenetic tree, two of these clusters together with previous Brazilian σC ARV sequences, and one cluster (VII) that was novel for Brazilian strains. Antigenic analysis showed that there were amino acids within putative epitopes located on the surface of the receptor-binding region of the σC protein with a high degree of variability.4. The study confirmed the presence of ARV genetic variants circulating in commercial birds in Brazil, and according to the antigenic prediction, the possibility of antigenic variants appears to be high.

Genotypic characterization and molecular evolution of avian reovirus in poultry flocks from Brazil.

Avian reovirus (ARV) is one of the main causes of infectious arthritis/tenosynovitis and malabsorption syndrome (MAS) in poultry. ARVs have been disseminated in Brazilian poultry flocks in the last years. This study aimed to genotype ARVs and to evaluate the molecular evolution of the more frequent ARV lineages detected in Brazilian poultry-producing farms. A total of 100 poultry flocks with clinical signs of tenosynovitis/MAS, from all Brazilian poultry-producing regions were positive for ARV by PCR. Seventeen bird tissues were submitted to cell culture and ARV RNA detection/genotyping by two PCRs. The phylogenetic classification was based on σC gene alignment using a dataset with other Brazilian and worldwide ARVs sequences. ARVs were specifically detected by both PCRs from the 17 cell cultures, and σC gene partial fragments were sequenced. All these sequences were aligned with a total of 451 ARV σC gene data available in GenBank. Phylogenetic analysis demonstrated five well-defined clusters that were classified into lineages I, II, III, IV, and V. Three lineages could be further divided into sub-lineages: I (I vaccine, Ia, Ib), II (IIa, IIb, IIc) and IV (IVa and IVb). Brazilian ARVs were from four lineages/sub-lineages: Ib (48.2%), IIb (22.2%), III (3.7%) and V (25.9%). The Bayesian analysis demonstrated that the most frequent sub-lineage Ib emerged in the world around 1968 and it was introduced into Brazil in 2010, with increasing spread soon after. In conclusion, four different ARV lineages are circulating in Brazilian poultry flocks, all associated with clinical diseases. RESEARCH HIGHLIGHTS One-hundred ARV-positive flocks were detected in all main poultry-producing regions from Brazil. A large dataset of 468 S1 sequences was constructed and divided ARVs into five lineages. Four lineages/sub-lineages (Ib, IIb, III and V) were detected in commercial poultry flocks from Brazil. Brazilian lineages shared a low identity with the commercial vaccine lineage (I vaccine). Sub-lineage Ib emerged around 1968 and was introduced into Brazil in 2010.

Chitosan-Based Delivery of Avian Reovirus Fusogenic Protein p10 Gene: In Vitro and In Vivo Studies towards a New Vaccine against Melanoma.

Reovirus is known to have an anticancer effect in both the preclinical and clinical assays. Current evidence suggests that the reovirus-mediated impact on tumor growth depends on the activation of specific antitumor immune responses. A feasible explanation for the oncolytic effects and immune system activation is through the expression of the fusogenic reovirus protein. In this work, we evaluated the in vivo antitumor effects of the expression of fusogenic protein p10 of avian reovirus (ARV-p10). We used chitosan nanoparticles (CH-NPs) as a vehicle for the ARV-p10 DNA in murine B16 melanoma models both in vitro and in vivo. We confirmed that ARV-p10 delivery through a chitosan-based formulation (ARV-p10 CH-NPs) was capable of inducing cell fusion in cultured melanoma cells, showing a mild cytotoxic effect. Interestingly, intratumor injection of ARV-p10 CH-NPs delayed tumor growth, without changing lymphoid populations in the tumor tissue and spleen. The injection of chitosan nanoparticles (CH-NPs) also delayed tumor growth, suggesting the nanoparticle itself would attack tumor cells. In conclusion, we proved that in vitro ARV-p10 protein expression using CH-NPs in murine melanoma cells induces a cytotoxic effect associated with its cell fusion. Further studies are necessary for establishing a protocol for efficient in vivo DNA delivery of fusion proteins to produce an antitumoral effect.

Molecular detection of Mycoplasma synoviae and avian reovirus infection in arthritis and tenosynovitis lesions of broiler and breeder chickens in Santa Catarina State, Brazil.

Infectious arthritis or tenosynovitis in broiler and breeder chickens results in major loss of productivity because of reduced growth and downgrading at processing plants. The most common causative agents of avian infectious arthritis are the bacterium Mycoplasma synoviae and avian reoviruses (ARVs) (family Reoviridae, genus Orthoreovirus). In this study, we evaluated the occurrence of these two pathogens in arthritis or tenosynovitis lesions of broilers and breeder flocks in southern Brazil using molecular detection. Tissue sections from tibiotarsal joints with visible lesions from 719 broilers and 505 breeders were analysed using pathogen-specific polymerase chain reaction (PCR) assays. In breeders, 41.2% (n = 296) of lesions were positive for M. synoviae, 26.4% (n = 190) were positive for ARV, while co-infection was present in 12.2% (n = 88) of the samples. In broilers, 20.8% (n = 105) of lesions were positive for M. synoviae, 11.9% (n = 60) for ARV and 7.7% (n = 39) of these cases were positive for both pathogens. Post-mortem examination revealed lesions with varying degrees of gross pathological severity. Histopathological examination showed intense, diffuse lymphohistiocytic inflammatory infiltrates with heterophil accumulation, primarily in the synovial capsule and digital flexor tendon, in all samples. Improved strategies for early detection and control of these major avian pathogens are highly desirable for preventing the spread of infection and reducing economic losses in the poultry industry.

Pathological and molecular findings of avian reoviruses from clinical cases of tenosynovitis in poultry flocks from Brazil.

Avian reoviruses (ARVs) can infect a variety of species worldwide. Birds can present stunting syndrome, respiratory and/or enteric diseases, immunosuppression, malabsorption, viral arthritis/tenosynovitis, and even secondary infections by other microorganisms. Flaws in conventional vaccines and the increase in the diagnostic rate of disease in the last 5 yr suggest the emergence of pathogenic ARVs in the poultry flocks worldwide. This study aimed to characterize birds lesions and to detect/genotype ARVs from a severe outbreak of tenosynovitis in broiler poultry flocks from Brazil. Tissue samples of lesions on pelvic limbs of broiler chicken were collected in poultry flocks with a high condemnation rate of carcasses due to lesions and submitted to histological and molecular analysis. Major gross pathological lesions included marked swelling, edema, and hemorrhages. Serous exudate was present between the tendons and hock joint. Histological examination demonstrated necrosis and inflammation of muscle fibers, mixed inflammatory infiltrate was observed in subcutaneous tissue and tendon sheaths. ARVs RNA was detected in 5 samples tested by polymerase chain reaction. These samples were also genotyped and demonstrated the occurrence of strains of the ARVs lineages II and V in the flocks. These results suggest that theses field ARVs, genetically distant from previously characterized strains, are associated to tenosynovitis and present in commercial Brazilian poultry flocks.

Distribution of IFITM3 in yellow-feathered broilers and inhibition of Avian Reovirus multiplication by IFITM3

This study was carried to express the interferon-induced transmembrane protein 3 (IFITM3) in vitro and examine its function in inhibition of avian reovirus (ARV) replication. The recombinant prokaryotic vector expressing yellow-feathered broiler IFITM3 was successfully constructed, and the recombinant protein was expressed in competent Escherichia coli BL21 cells. New Zealand white rabbits were immunized with the purified recombinant protein to prepare a polyclonal antibody, with a titer of 1:128,000. Immunohistochemistry, reverse transcription-PCR, and real-time fluorescence quantitative PCR showed that IFITM3 was distributed in the yellow-feathered broiler immune organs, and the expression of IFITM3 in bursa of Fabricius was more than in spleen and thymus. It was found that in the thymus, spleen and bursa of Fabricius the mRNA expression levels of IFN and IFITM3 were significantly induced after ARV infection. And it was also certified in the chicken embryo fibroblasts (CEFs) which infected with ARV. Then the IFN was added into the cell culture medium before CEFs were infected with ARV. The results indicated that the mRNA of IFITM3 expression was significantly increased and ARV multiplication was significantly inhibited. And when the expression of IFITM3 was knocked down by siRNA-IFITM3, the expression of IFITM3 was significantly reduced, but the ARV multiplication was significantly increased, which indicated that IFITM3 protein could inhibit the ARV replication.

Distribution of IFITM3 in yellow-feathered broilers and inhibition of Avian Reovirus multiplication by IFITM3

This study was carried to express the interferon-induced transmembrane protein 3 (IFITM3) in vitro and examine its function in inhibition of avian reovirus (ARV) replication. The recombinant prokaryotic vector expressing yellow-feathered broiler IFITM3 was successfully constructed, and the recombinant protein was expressed in competent Escherichia coli BL21 cells. New Zealand white rabbits were immunized with the purified recombinant protein to prepare a polyclonal antibody, with a titer of 1:128,000. Immunohistochemistry, reverse transcription-PCR, and real-time fluorescence quantitative PCR showed that IFITM3 was distributed in the yellow-feathered broiler immune organs, and the expression of IFITM3 in bursa of Fabricius was more than in spleen and thymus. It was found that in the thymus, spleen and bursa of Fabricius the mRNA expression levels of IFN and IFITM3 were significantly induced after ARV infection. And it was also certified in the chicken embryo fibroblasts (CEFs) which infected with ARV. Then the IFN was added into the cell culture medium before CEFs were infected with ARV. The results indicated that the mRNA of IFITM3 expression was significantly increased and ARV multiplication was significantly inhibited. And when the expression of IFITM3 was knocked down by siRNA-IFITM3, the expression of IFITM3 was significantly reduced, but the ARV multiplication was significantly increased, which indicated that IFITM3 protein could inhibit the ARV replication.(AU)

Investigation of enteric viruses in the feces of neotropical migratory birds captured on the coast of the State of Pará, Brazil

Migratory birds can become long-distance vectors for a wide range of microorganisms and can cause human disease, being the Brazilian coast a gateway for northern migratory birds. These animals are considered natural reservoirs of different viruses that cause important diseases, being relevant research of viral pathogens in migratory birds to epidemiology surveillance. The objective of the study was to investigate the presence of avian rotavirus (AvRV), avian reovirus (ARV) and picobirnavirus (PBV) in Neotropical migratory birds captured on the coast of Brazil. A total of 23 individual fecal samples of the migratory birds species Calidris pusilla (20 birds), Numenius phaeopus (1 bird) and Charadrius semipalmatus (2 birds) were collected. Fecal suspensions were prepared from the collected samples for subsequent extraction of double-stranded RNA (dsRNA), which was subjected to polyacrylamide gel electrophoresis (PAGE) and reverse transcription polymerase chain reaction (RT-PCR). The electrophoretic profiles were not detected by PAGE, and the amplification for the studied viruses PBV, ARV and AvRV (specie D, gene VP6 and NSP4) were negative. Positivity for AvRVD, VP7 gene was of 4.35% (1/23) for the migratory bird Calidris pusilla. After sequencing and building the tree of phylogenetic relationships avian Rotavirus Group D identified in this study was phylogenetically related and grouped into one branch, together to previously reported AvRVD from Brazil in chicken flocks with 99.8% nucleotide and 100% amino acid similarities.(AU)

Investigation of enteric viruses in the feces of neotropical migratory birds captured on the coast of the State of Pará, Brazil

Migratory birds can become long-distance vectors for a wide range of microorganisms and can cause human disease, being the Brazilian coast a gateway for northern migratory birds. These animals are considered natural reservoirs of different viruses that cause important diseases, being relevant research of viral pathogens in migratory birds to epidemiology surveillance. The objective of the study was to investigate the presence of avian rotavirus (AvRV), avian reovirus (ARV) and picobirnavirus (PBV) in Neotropical migratory birds captured on the coast of Brazil. A total of 23 individual fecal samples of the migratory birds species Calidris pusilla (20 birds), Numenius phaeopus (1 bird) and Charadrius semipalmatus (2 birds) were collected. Fecal suspensions were prepared from the collected samples for subsequent extraction of double-stranded RNA (dsRNA), which was subjected to polyacrylamide gel electrophoresis (PAGE) and reverse transcription polymerase chain reaction (RT-PCR). The electrophoretic profiles were not detected by PAGE, and the amplification for the studied viruses PBV, ARV and AvRV (specie D, gene VP6 and NSP4) were negative. Positivity for AvRVD, VP7 gene was of 4.35% (1/23) for the migratory bird Calidris pusilla. After sequencing and building the tree of phylogenetic relationships avian Rotavirus Group D identified in this study was phylogenetically related and grouped into one branch, together to previously reported AvRVD from Brazil in chicken flocks with 99.8% nucleotide and 100% amino acid similarities.

Avian reovirus infection: an overview / Reovirose aviária: um panorama

Poultry production is an activity of great importance in Brazilian economy, both due to the domestic consumption and the large amount of chicken meat exportation. Poultry activity modernization allowed the creation of animals in high density facilities, however, it facilitates the rapid dissemination of pathogens, which reduces the productivity rates. This review aims to highlight the avian reovirus, an important agent of arthritis in birds that has a worldwide distribution. The affected birds present a reduction in weight gain due to movement difficulties. In addition to arthritis, the virus may be related to a variety of pathological conditions, such as enteric and respiratory disorders, Hepatitis and myocarditis. The main prevention and control measure is the flock vaccination. Nevertheless, due to the avian reovirus great genetic variability, the vaccine may not be effective against circulating strains. This article aims to overview the virus biology, its variability and classification, and the infection pathology and diagnosis.(AU)

A avicultura é um setor de grande importância na economia brasileira tanto pelo aumento do consumo interno quanto pelo crescimento na exportação de carne de frango. A modernização da atividade avícola permitiu a criação adensada de animais, facilitando, no entanto, a rápida disseminação de patógenos que reduzem os índices de produtividade dos plantéis. Nesta revisão, é destacado o reovírus aviário, importante agente de artrite em aves que apresenta distribuição mundial. As aves acometidas apresentam redução no ganho de peso devido à dificuldade de locomoção. Além da artrite, o vírus pode estar relacionado a uma variedade de condições patológicas, como distúrbios entéricos e respiratórios, hepatite e miocardite. A principal forma de prevenção e controle é a vacinação do plantel. No entanto, devido à grande variabilidade genética do reovírus aviário, a vacina utilizada pode não ser eficiente contra estirpes que circulam no campo. O artigo traz uma visão geral sobre a biologia do vírus, sua variabilidade e propostas de classificação dos isolados, patologia da doença e diagnóstico da infecção.(AU)

Avian reovirus infection: an overview / Reovirose aviária: um panorama

Poultry production is an activity of great importance in Brazilian economy, both due to the domestic consumption and the large amount of chicken meat exportation. Poultry activity modernization allowed the creation of animals in high density facilities, however, it facilitates the rapid dissemination of pathogens, which reduces the productivity rates. This review aims to highlight the avian reovirus, an important agent of arthritis in birds that has a worldwide distribution. The affected birds present a reduction in weight gain due to movement difficulties. In addition to arthritis, the virus may be related to a variety of pathological conditions, such as enteric and respiratory disorders, Hepatitis and myocarditis. The main prevention and control measure is the flock vaccination. Nevertheless, due to the avian reovirus great genetic variability, the vaccine may not be effective against circulating strains. This article aims to overview the virus biology, its variability and classification, and the infection pathology and diagnosis.

A avicultura é um setor de grande importância na economia brasileira tanto pelo aumento do consumo interno quanto pelo crescimento na exportação de carne de frango. A modernização da atividade avícola permitiu a criação adensada de animais, facilitando, no entanto, a rápida disseminação de patógenos que reduzem os índices de produtividade dos plantéis. Nesta revisão, é destacado o reovírus aviário, importante agente de artrite em aves que apresenta distribuição mundial. As aves acometidas apresentam redução no ganho de peso devido à dificuldade de locomoção. Além da artrite, o vírus pode estar relacionado a uma variedade de condições patológicas, como distúrbios entéricos e respiratórios, hepatite e miocardite. A principal forma de prevenção e controle é a vacinação do plantel. No entanto, devido à grande variabilidade genética do reovírus aviário, a vacina utilizada pode não ser eficiente contra estirpes que circulam no campo. O artigo traz uma visão geral sobre a biologia do vírus, sua variabilidade e propostas de classificação dos isolados, patologia da doença e diagnóstico da infecção.

The occurrence of orthoreovirus, rotavirus and chicken anemia virus in chickens of the poultry industry in Minas Gerais, Brazil / Ocorrência de orthoreovirus, rotavirus e vírus da anemia das galinhas em frangos de corte da indústria avícola de Minas Gerais

Fifty-four fecal samples taken from broiler chickens from 1 to 45 days of age, and of pullets from 10 to 13 weeks of age, original from eight different poultry regions in the state of Minas Gerais, Brazil, were collected from March 2008 to January 2010 for avian Orthoreovirus (ARV) and avian Rotavirus (AvRV) analyses. For the assay of ARV, RNA was immediately extracted (Trizolâ) and transcribed into cDNA for assaying in a nested-PCR with ARV-specific primers. For AvRV, polyacrylamide gel electrophoresis (PAGE) was performed with RNA extracts obtained by phenol-chloroform extraction. CAV was additionally investigated through a nested-PCR of thymus and spleen. Results found 5.55% positive for ARV and 9.25% for AvRV. Also, CAV and ARV genomes were detected in co-infection, in a highly prostrated and claudicating chicken flock. No ARV or AvRV infections were detected in pullets. Material of a clinically affected flock was inoculated into SPF embryos, resulting in embryonic hemorrhage, whitish foci in the chorio-allantoic membrane and death. Sequencing of ARV amplicons and isolate cDNA grouped local strains with the ARV S1133 strain, historically used in live vaccines, suggesting the continued circulation of this vaccine virus strain in intensive poultry regions. Detection rates for ARV and AvRV, as well as the presence of CAV, were additionally indicative of failing biosecurity strategies for the intensive poultry regions examined.

Avaliou-se a ocorrência de Orthoreovirus (ARV) e Rotavirus (AvRV) aviários na avicultura industrial de Minas Gerais. Foram colhidas cinquenta e quatro amostras de fezes de frangos de corte entre um e 45 dias e de frangas de postura de 10 a 13 semanas de idade. Para análise de ARV, o RNA foi imediatamente extraído (Trizol), transcrito em cDNA e avaliado em uma PCR com oligonucleotídeos iniciadores específicos para ARV. Para a investigação de AvRV, os extratos de RNA foram obtidos por fenol-clorofórmio e submetidos à eletroforese em gel de poliacrilamida. Todas as amostras foram também avaliadas para o DNA do vírus da anemia das galinhas (CAV) em uma nested-PCR específica. Em frangos de corte, a positividade encontrada para ARV foi de 5,55% e para AvRV de 9,25%. CAV foi detectado em coinfecção em um plantel com refugagem, claudicação e prostração. Nenhuma amostra de poedeiras foi positiva para ARV ou AvRV. Material de plantel com sinais clínicos foi purificado e inoculado em ovos SPF embrionados, sendo obtidas lesões hemorrágicas e focos brancos na membrana cório-alantóide. O sequenciamento dos produtos de PCR e de embrião agrupou os isolados de ARV com a estirpe S1133, historicamente usada como vacina viva. Os resultados sugerem a continuada circulação da infecção por estirpes assemelhadas a ARV S1133 nas regiões de avicultura industrial. Os índices de detecção de ARV, AvRV e CAV indicam que a intensificação nas regiões produtoras tem resultado em falhas de biosseguridade.

A retrospective PCR investigation of avian Orthoreovirus, chicken infectious anemia and fowl Aviadenovirus genomes contamination in commercial poultry vaccines in Brazil / Estudo retrospectivo de vacinas avícolas vivas por PCR para os vírus da anemia das galinhas, Aviadenovirus aviário e Orthoreovirus aviário

Vacinas avícolas vivas comerciais produzidas entre 1991 e 2005 foram examinadas para a presença de genomas dos vírus da anemia infecciosa das galinhas (Gyrovirus CAV), da hepatite por corpúsculo de inclusão (Aviadenovirus FAdV) e da artrite viral/síndrome da má absorção (Orthoreovirus aviário ARV). Vinte e seis partidas de vacinas vivas liofilizadas de oito fabricantes com lacre original foram examinadas. As extrações de DNA e PCR de CAV e FAdV, e de RNA e RT-PCR para ARV, foram descritas previamente. Contaminações triplas de ARV, CAV e FAdV foram detectadas em vacinas de mesmo fabricante, produzidas em 1991 e 1992 contra a doença de Newcastle (DN), e para a encefalomielite aviária, produzida em 1994. ARV e CAV em co-infecção foram encontrados em vacinas contra a doença de Marek liofilizadas produzidas em 1996 por dois fabricantes diferentes. Genoma de ARV foi detectado em vacinas contra a bronquite infecciosa de setembro e dezembro de 1998, doença infecciosa bursal, de dezembro de 1998 e DN de janeiro de 1998. Três dos oito fabricantes apresentaram vacinas com contaminação e cinco nunca apresentaram vacinas contaminadas. Nenhuma vacina produzida a partir de 2001 apresentou contaminação. Cogita-se um papel epidemiológico para vacinas vivas, como fonte de infecção para ARV, CAV e FAdV e, potencialmente determinante da atual alta disseminação destes.(AU)

The occurrence of Orthoreovirus, Rotavirus and chicken anemia virus in chickens of the poultry industry in Minas Gerais, Brazil / Ocorrência de Orthoreovirus, Rotavirus e vírus da anemia das galinhas em frangos de corte da indústria avícola de Minas Gerais

Fifty-four fecal samples taken from broiler chickens from 1 to 45 days of age, and of pullets from 10 to 13 weeks of age, original from eight different poultry regions in the state of Minas Gerais, Brazil, were collected from March 2008 to January 2010 for avian Orthoreovirus (ARV) and avian Rotavirus (AvRV) analyses. For the assay of ARV, RNA was immediately extracted (Trizolâ) and transcribed into cDNA for assaying in a nested-PCR with ARV-specific primers. For AvRV, polyacrylamide gel electrophoresis (PAGE) was performed with RNA extracts obtained by phenol-chloroform extraction. CAV was additionally investigated through a nested-PCR of thymus and spleen. Results found 5.55% positive for ARV and 9.25% for AvRV. Also, CAV and ARV genomes were detected in co-infection, in a highly prostrated and claudicating chicken flock. No ARV or AvRV infections were detected in pullets. Material of a clinically affected flock was inoculated into SPF embryos, resulting in embryonic hemorrhage, whitish foci in the chorio-allantoic membrane and death. Sequencing of ARV amplicons and isolate cDNA grouped local strains with the ARV S1133 strain, historically used in live vaccines, suggesting the continued circulation of this vaccine virus strain in intensive poultry regions. Detection rates for ARV and AvRV, as well as the presence of CAV, were additionally indicative of failing biosecurity strategies for the intensive poultry regions examined.(AU)

Avaliou-se a ocorrência de Orthoreovirus (ARV) e Rotavirus (AvRV) aviários na avicultura industrial de Minas Gerais. Foram colhidas cinquenta e quatro amostras de fezes de frangos de corte entre um e 45 dias e de frangas de postura de 10 a 13 semanas de idade. Para análise de ARV, o RNA foi imediatamente extraído (Trizol), transcrito em cDNA e avaliado em uma PCR com oligonucleotídeos iniciadores específicos para ARV. Para a investigação de AvRV, os extratos de RNA foram obtidos por fenol-clorofórmio e submetidos à eletroforese em gel de poliacrilamida. Todas as amostras foram também avaliadas para o DNA do vírus da anemia das galinhas (CAV) em uma nested-PCR específica. Em frangos de corte, a positividade encontrada para ARV foi de 5,55% e para AvRV de 9,25%. CAV foi detectado em coinfecção em um plantel com refugagem, claudicação e prostração. Nenhuma amostra de poedeiras foi positiva para ARV ou AvRV. Material de plantel com sinais clínicos foi purificado e inoculado em ovos SPF embrionados, sendo obtidas lesões hemorrágicas e focos brancos na membrana cório-alantóide. O sequenciamento dos produtos de PCR e de embrião agrupou os isolados de ARV com a estirpe S1133, historicamente usada como vacina viva. Os resultados sugerem a continuada circulação da infecção por estirpes assemelhadas a ARV S1133 nas regiões de avicultura industrial. Os índices de detecção de ARV, AvRV e CAV indicam que a intensificação nas regiões produtoras tem resultado em falhas de biosseguridade.(AU)

Anticorpos contra vírus em galinhas de terreiro do Estado do Rio Grande do Sul, Brasil / Antibodies against avian viruses in backyard poultry from the Rio Grande do Sul State, Brazil

No Brasil, a população de aves conhecida como galinhas de terreiro encontra-se fora do sistema de biosseguridade aplicada às criações comerciais. Para investigar a presença de anticorpos contra alguns vírus específicos nesta população, foram coletadas amostras de sangue de 867 aves não-vacinadas em 60 propriedades de 22 municípios do Estado do Rio Grande do Sul, Brasil. O soro foi testado para a presença de anticorpos contra o vírus da bronquite infecciosa das galinhas (IBV), reovírus aviário (ARV) e o vírus da doença infecciosa da bolsa (IBDV) pela técnica de soroneutralização. Anticorpos contra IBV foram detectados em 65 por cento (564/867) das amostras, contra ARV em 21,6 por cento (187/867) e contra IBDV em 80,2 por cento (695/867) das aves. Todas as propriedades avaliadas apresentavam uma ave positiva para anticorpos contra IBV e IBDV e 88,3 por cento delas eram positivas para ARV. Os resultados demonstram que esses vírus estão presentes em galinhas de terreiro nas criações avícolas não-industriais da região central do Estado. Os resultados indicam a necessidade de um programa de vigilância permanente nessa população e ainda indicam a necessidade de avaliar o impacto destas infecções nos próprios plantéis e o risco associado à transmissão destas às criações comerciais.

The backyard poultry are not included in the biosecurity system applied in commercial flocks in Brazil. To investigate the presence of antibodies to specific viral pathogens in this population, blood samples were collected from 867 non-vaccinated birds, from 60 flocks in 22 counties of the Rio Grande do Sul State, Brazil. The samples were tested to detect antibodies against infectious bronchitis virus (IBV), avian reovirus (ARV) and infectious bursal disease virus (IBDV), through the virus neutralization test. Antibodies to IBV were detected in 65 percent (564/867), against ARV in 21.6 percent (187/867), and against IBDV in 80.2 percent (695/867) of the samples. All the flocks had chickens positive to IBV and IBDV antibodies, and 88.3 percent of them harbored antibodies to ARV. The results show the presence of these viruses in backyard poultry from the central region of the State. It also indicates the need for additional studies aimed to evaluate the real importance of these infections for this type of flocks.

Segmented double-stranded genomic RNA viruses in fecal samples from broiler chicken

A técnica de eletroforese em gel de poliacrilamida foi utilizada com o objetivo de identificar vírus com genoma contituído por RNA de fita dupla (dsRNA) segmentado, em material fecal de frangos de corte. Foram analisadas 378 amostras de fezes de aves, com idade entre a primeira e sétima semanas de vida, provenientes de granjas avícolas localizadas no Estado do Paraná, Brasil. dsRNA com perfil de migracão característico de rotavírus (AvRV), reovírus (ARV) ou picobirnavírus (PBV), foi identificado em 32 (8,5 per center), 7 (1,8 per center) e 13 (3,4 per center) amostras, respectivamente. AvRV e ARV ocorreram com maior freqüência em aves com até um mês de idade e estiveram diretamente relacionados a fezes diarréicas e pastosas, provenientes de aves com sinais clínicos de enterite. Considerando-se apenas as amostras de fezes colhidas em aves com diarréia, o AvRV foi detectado em 37,8 per center (14/37) e o ARV em 13,5 per center (5/37) da amostragem analisada. Em fezes com aspectos normais (controle) obtidas de aves clinicamente sadias, o AvRV foi identificado em apenas 1,5 per center (4/274) e o ARV não foi detectado. O ácido nucléico do PBV foi detectado com maior freqüência em fezes pastosas colhidas de aves com duas a sete semanas de vida. O AvRV apresentou grande variabilidade eletroforética dos segmentos de dsRNA, tendo sido identificados nove eletroferotipos distintos. Não foram observadas variacões no perfil genômico nas amostras de ARV e também de PBV.

Lesöes articulares em frangos de corte (Gallus gallus) na infecçäo experimental pelo reovírus aviário / Articular lesion in experimental infeccion with avian reovirus in broiler (Gallus gallus)

Este trabalho descreve as lesöes articulares induzidas pela inoculaçäo oral e podal de uma amostra de reovírus aviário (S-1133) em pintos de corte de um dia de idade. Realizou-se o exame histopatológico de fragmentos da articulaçäo tibiotársica utilizando-se cinco aves dos grupos inoculados e grupo controle nos períodos de 24, 48, 72 horas e semanalmente até a oitava semana após a inoculaçäo. A primeira alteraçäo observada foi um infiltrado inflamatório misto nas bainhas tendinosas uma semana após a inoculaçäo. Na segunda semana após a inoculaçäo, houve fibroplasia, formaçäo de folículos linfóides e hiperplasia das células da membrana sinovial, alteraçöes progressivas observadas até o final do período experimental. As lesöes articulares foram similares, ocorrendo simultaneamente nos dois grupos inoculados, contudo as alteraçöes foram mais severas no grupo inoculado no coxim plantar

Lesöes viscerais induzidas experimentalmente pela inoculaçäo de uma amostra artrotrópica de reovírus em frangos de corte (Gallus gallus) / Visceral lesion induced experimentally by an artrotropic strain of reovirus in broiler (Gallus gallus)

Este trabalho descreve as lesöes viscerais induzidas pela inoculaçäo oral e podal de uma amostra artrotrópica de reovírus aviário (S-1133) em pintos de corte de um dia de idade. Realizou-se o exame histopatológico de fragmentos da Bursa de Fabricius, intestinos, fígado, baço e miocárdio utilizando-se cinco aves dos grupos inoculados e do grupo controle, nos períodos de 24, 48, 72 horas e semanalmente até a oitava semana após a inoculaçäo. No miocárdio, observou-se miocardite intersticial 48 horas pós-inoculaçäo, miocardite necrótica e pericardite na primeira semana após a inoculaçäo. Houve no pericárdio, na segunda semana após a inoculaçäo, a formaçäo de folículos linfóides e seu espessamento fibroso em períodos subseqüentes até a oitava semana. No fígado, foi observado um infiltrado inflamatório misto 72 horas após a inoculaçäo e degeneraçäo vacuolar e necrose dos hepatócitos na primeira semana pós-inoculaçäo. Observou-se, ainda, a presença de tecido linfóide ativo e de infiltrado heterofílico na segunda semana após a inoculaçäo, persistindo até o final do experimento. Verificou-se necrose progressiva do tecido esplênico de 48 horas até a primeira semana após a inoculaçäo e a partir da segunda semana observou-se hiperplasia dos folículos linfóides esplênicos. Os demais órgäos examinados näo apresentaram alteraçöes. As lesöes viscerais foram similares ocorrendo simultaneamente nos dois grupos inoculados, contudo as alteraçöes foram mais severas no grupo inoculado no coxim plantar