Detection of infectious bronchitis virus and specific anti-viral antibodies using a concanavalin A-Sandwich-ELISA

Concanavalin A-Sandwich ELISA (Con A-S-ELISA) was developed for the detection of infectious bronchitis virus (IBV) or chicken specific anti-viral antibodies. The antigen detection limit for the Con A-S-ELISA was 10(5,1) EID50/mL. Three homologous and four heterologous IBV strains were similarly detected. This assay was highly effective in detecting the virus after infected tissue homogenates were passed once in embryonated chicken eggs, showing a good agreement with virus isolation technique. The Con A-S-ELISA was also used to measure anti-IBV chicken antibodies and showed a high coefficient of correlation (r = 0.85) and an agreement of k = 0.80 with the commercially available Indirect-ELISA. The relative sensitivity and specificity between these two tests were, respectively, 92.86% and 95.65% with an accuracy of 93.39%. Thus, the Con A-S-ELISA proved to be able to detect alternatively homologous and heterologous IBV strains or specific chicken anti-IBV antibodies, using the Con A as capture reagent of this assay.

Produtividade e qualidade dos frutos da laranjeira 'Pêra' clone IAC em 16 porta-enxertos na região de Bebedouro-SP

Avaliou-se a influência de 16 porta-enxertos na produtividade, nas características físicas e químicas (sólidos solúveis totais-°Brix; acidez; ratio; porcentagem de suco; índice tecnológico e tamanho dos frutos) dos frutos da laranjeira 'Pêra' [Citrus sinensis (L.) Osbeck] e na incidência e severidade da clorose variegada dos citros (CVC). O plantio do experimento foi realizado em julho de 1993, com espaçamento de 6,0 m entre linhas e 3,5 m entre plantas (476 plantas/ha). O experimento foi conduzido sem irrigação. O delineamento experimental foi em blocos ao acaso, duas plantas por parcela, três repetições e 16 tratamentos, constituídos pelas seguintes cultivares porta-enxertos: tangerineira 'Sun Chu Sha Kat' (Citrus reticulata Blanco), tangerineira 'Pectinífera' (C. reticulata), 'Shekwasha' (C. depressa Hayata), tangerineira 'Pectinífera/Shekwasha' (C. depressa Hayata), tangerineira 'Batangas' (C. reticulata), tangerineira 'Oneco' (C. reticulata), citrangor [citrange (Poncirus trifoliata Raf. x C. sinensis) x C. sinensis], citrandarin [C.sunki hort. Ex Tanaka) x Poncirus trifoliata (L.) Raf. cv. English, tangerineira 'Sunki' (C. sunki), tangerineira 'Suen-Kat' (C. sunki), tangerineira 'Nasnaran' (C. amblycarpa Ochse), tangerineira 'Venezuela' (C. reticulata), tangerineira Heen Naran (C. lycopersicaeformis hort. ex Tan. ), limoeiro 'Cravo' (C. limonia Osbeck) x tangerineira 'Cleópatra' (C. reshni hort ex Tanaka), limoeiro 'Cravo' (C. limonia), tangerineira 'Cleópatra' (C. reshni). A intensidade da clorose variegada dos citros variou em função dos porta-enxertos e não se relacionou com a produção de frutos até a quarta safra. Os porta-enxertos estudados, com exceção da tangerineira Nasnaran, proporcionaram qualidade e produções iniciais de frutos similares aos do limoeiro 'Cravo'.

Development and application of a Saccharomyces cerevisiae-expressed nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibodies against infectious bronchitis virus

A Saccharomyces cerevisiae-expressed nucleocapsid (N) polypeptide of the M41 strain of infectious bronchitis virus (IBV) was used as antigen in a recombinant yeast-expressed N protein-based enzyme-linked immunosorbent assay (Y-N-ELISA). The Y-N-ELISA was rapid, sensitive, and specific for detecting chicken serum antibodies to IBV, and it compared favorably with a commercial ELISA.

Infectious bronchitis virus: detection and vaccine Strain differentiation by semi-nested RT-PCR

A semi-nested reverse transcription-polymerase chain reaction (Semi-N-RT-PCR) was developed and used to detect the S glycoprotein gene of infectious bronchitis virus (IBV) strains and to discriminate H120 vaccine strain from other strains. Viral RNA was extracted from the allantoic fluid of chicken embryos and from tissues of chickens experimentally infected with different strains of IBV. Amplification and identification of the viral RNA was performed using two sets of primers complementary to a region of the S glycoprotein gene in the Semi-N-RT-PCR assay. The pair of primers used in the first PCR consisted of universal oligonucleotides flanking a more variable region of S1-S2 gene. The second primer pair was used in the Semi-N-RT-PCR and was comprised of one of the primers from the first universal pair together with either another universal internal oligolucleotide or a oligonucleotide sequence specific for the H120 strain of IBV. The universal primers detected all reference IBV strains and field isolates tested herein. The Semi-N-RT-PCR had high sensitivity and specificity, and was able to differentiate the H120 vaccine strain from other reference IBV strains; including M41 strain. All tissue samples collected from chickens experimentally infected with H120 or M41 strains were positive in the semi-nested RT-PCR using universal primers, while only the H120-infected tissue samples were amplified by the set of primers containing the H120-oligonucleotide. In conclusion, the ability of Semi-N-RT-PCR to detect distinct IBV strains and preliminarily discriminate the vaccine strain (H120) closes a diagnostic gap and offers the opportunity to use comprehensive PCR procedures for the IBV diagnosis.

Effect of mannanoligosaccharides and/or enzymes on antibody titers against infectious bursal and Newcastle disease viruses

O efeito da inclusão de mananoligossacarídeo (MOS) e/ou enzimas em dietas de frangos sobre os títulos de anticorpos contra os vírus das doenças de Gumboro (VDG) e de Newcastle (VDN). Setecentos e cinqüenta aves foram distribuídas em um delineamento experimental inteiramente ao acaso, em arranjo fatorial 2 x 2 + 1, com dois níveis de MOS (0 e 0,1% até 21 dias e 0,05% de 22 até 42 dias de idade), dois níveis de enzimas (0 e 0,05%) e uma dieta-controle-positivo contendo antibióticos, totalizando cinco tratamentos com cinco repetições. Para análise dos anticorpos, amostras de sangue foram colhidas semanalmente por punção da veia jugular em duas aves de cada repetição. A primeira e a última colheita foram realizadas aos sete e 42 dias de idade, respectivamente. A inclusão de MOS resultou em aumento dos títulos contra VDG na quarta (P<0,03) e quinta (P<0,02) semanas, e contra VDN na terceira (P<0,01), quarta (P<0,03) e quinta (P<0,03) semanas de idade. O MOS foi efetivo em estimular a resposta imune humoral contra VDG e VDN vacinais.

A double antibody sandwich ELISA for rapid diagnosis of virus infection and to measure the humoral response against infectious bursal disease on clinical material

A double antibody sandwich ELISA (DAS-ELISA) was developed and employed for simultaneous direct detection of infectious bursal disease virus (IBDV) from bursal samples and to measure the humoral response, using the same basic immunoreagents, the purified and non-purified antigen, capture antibody and chicken hyperimmune sera were prepared, and standardized for this purpose, the DAS-ELISA was applied to both 80 bursal suspensions and 224 corresponding serum samples from vaccinated and non-vaccinated commercial hocks, Bursae samples were collected at 2 weeks of age, and submitted to histological examination, virus isolation in specific pathogen-free chickens embryos, and the DAS-ELISA technique, Serum titres obtained in indirect ELISA and serum neutralization test were compared with those in DAS-ELISA, the agreement was 80% between DAS-ELISA, and the conventional techniques, with high sensitivity (87%) and specificity (90%).

Replication of classical infectious bursal disease virus in the chicken embryo related cell line

Infectious bursal disease (IBD) is an acute, highly contagious viral disease. The diagnosis of IBD depends on time-consuming and costly procedures, like virus isolation on chick embryos and histopathological examination, A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), immunoperoxidase and reverse transcription polymerase chain reaction (RT-PCR) were applied in this study to detect classical IBD virus (IBDV) after three blind passages of the Lukert strain on chicken embryo related (CER) cell monolayer after different periods of infection: 6, 12, 24 and 48 h, Cytophatic effects were most evident 12 h post-infection (p.i.) but were observed at 6 h p.i. The maximum discrimination between IBDV-infected and uninfected cell suspensions obtained by the use of DAS-ELISA for virus detection corresponded to 0.597+/-0.02 and 0.010+/-0.01 after 12h p.i., respectively. The RT-PCR was performed using the set of primers A3.1 and A3.2 to amplify the VP2 region of the IBDV genome, This molecular technique demonstrated that from 6 h p.i., it was possible to detect the viral RNA. The results show that the CER cell line can be used for classical IBDV propagation, confirmed by the DAS-ELISA, immunoperoxidase and RT-PCR assay.

Infectious bronchitis virus replication in the chicken embryo related cell line

The susceptibility of the chicken embryo related (CER) cell line to infectious bronchitis virus (IBV M41) was characterized after five consecutive passages in CER cells. Virus replication was monitored by cytopathic effect observation, electron microscopy, indirect immunofluorescence, and reverse transcription polymerase chain reaction (RT-PCR). At 96 h post-infection (p.i.), the cytopathic effect was graded 75% by cell fusion, rounding up of cells and monolayer detachment, and the electron microscopy image characterized by coronavirus morphology. Cytoplasmic fluorescence was readily observed by from 24 h p.i. onwards, and at all times the respective viral RNA from IBV-infected monolayers was demonstrated by RT-PCR. Extra-cellular virus was measured by virus titration performed on chicken kidney cells and embryonated chicken eggs, and respective titres ranged from 4.0 to 6.0 log(10) EID50/ml on embryonated chicken eggs, and from 2.0 to 6.0 log(10) TCID50/ml on both CER cells and chicken kidney cells studied from 24 to 120 h p.i. These results confirmed that the M41 strain replicated well in the CER cell line.

A liquid phase blocking ELISA for the detection of antibodies against infectious bronchitis virus

A liquid phase blocking ELISA (LPB-ELISA) was developed for the detection and measurement of antibodies against infectious bronchitis virus (IBV). The purified and nonpurified virus used as antigen, the capture and detector antibodies, and the chicken hyperimmune sera were prepared and standardized for this purpose. A total of 156 sera from vaccinated and 100 from specific pathogen-free chickens with no recorded contact with the virus were tested. The respective serum titers obtained in the serum neutralization test (SNT) were compared with those obtained in the LPB-ELISA. There was a high correlation (r2 = 0.8926) between the two tests. The LPB-ELISA represents a single test suitable for the rapid detection of antibodies against bronchitis virus in chicken sera, with good sensitivity (88%), specificity (100%) and agreement (95.31%).

Direct detection of infectious bursal disease virus from clinical samples by in situ reverse transcriptase-linked polymerase chain reaction

The presence of the very virulent (vv) Brazilian strain of infectious bursal disease virus (IBDV) was determined in the bursa of Fabricius, thymus and liver of 2-week-old broilers from a flock with a higher than expected mortality. For this purpose, a direct in situ reverse transcriptase (RT)-linked polymerase chain reaction (PCR) method was developed using specific primers for vvIBDV. Unlabelled forward and reverse biotinylated oligonucleotides were used for RT-PCR in a one-step method and the respective products were revealed by a direct enzymatic reaction. The results were compared with those obtained by standard RT-PCR using general primers for IBDV and virus isolation. The virus isolation, RT-PCR and in situ RT-PCR revealed positive results on the bursa of Fabricius in 86%, 80% and 100%, respectively. The in situ RT-PCR detected vvIBDV in all tested thymus and liver samples, whereas the standard RT-PCR detected virus in 80% and 90% of the samples, respectively. After three consecutive passages on chicken embryonated eggs, IBDV was isolated from 64% of the thymus samples and 30% of the liver samples. In the present study, no classical or antigenic variants of IBDV were detected. The developed in situ RT-PCR assay was able to detect the very virulent strain of IBDV with a higher sensitivity than the conventional RT-PCR and virus isolation.

Emerging infectious diseases in cetaceans worldwide and the possible role of environmental stressors

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular epidemiology and evolution of avian infectious bronchitis virus

Mutation and recombination processes are involved in the genetic and phenotypic variations of RNA viruses, leading to the emergence of new variant strains, and give rise to virus population diversity to be modeled by the host, particularly by the immune system, as occurred with infectious bronchitis virus (IBV) in chickens. The consequence is a continuous emergence of new IBV variants with regard to pathotypes, serotypes, and protectotypes. Nucleotide sequencing and subsequent genetic analysis of the S1 and N protein gene sequences provide a fast and accurate method to classify and predict IBV genotype, and a powerful instrument to monitor phylogenetic and epidemiological evolution of IBV variants. Despite the use of vaccination programmes, infectious bronchitis has become a serious problem in Brazil. Thus, a significant number of IBV field variants have been identified circulating in the Brazilian commercial poultries between 2000 to 2006 and more recently in Argentina. These viruses seem to be indigenous, because they demonstrated a low genetic relatedness with the majority of the reference strains from North America, Europe and Asia, but were moderately to highly related one to another. In summary, indigenous field IBV variants were evolving and circulating in the field in Brazil and Argentina, and should be considered as initial candidates for protection against current IBV infectious in chickens. However, in vitro and in vivo studies are needed to determine the pathogenicity and immunogenecity of these new isolates, before defining a new vaccine strain.

Plants of an Eucalyptus clone damaged by Scolytidae and Platypodidae (Coleoptera)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)