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.

Detection of turkey coronavirus in commercial turkey poults in Brazil

Poult enteritis complex has been incriminated as a major cause of loss among turkey poults in other countries. We have observed this in Brazil, associated with diarrhoea, loss of weight gain and, commonly, high mortality In this study, we have used the reverse transcriptase polymerase chain reaction (RT-PCR) to detect turkey coronavirus (TCoV) in sick poults 30 to 120 days of age from a particular producer region in Brazil. The RT-PCR was applied to extracts of intestine tissue suspensions, and the respective intestinal contents, bursa of Fabricius, faecal droppings and cloacal swabs. Primers were used to amplify the conserved 3' untranslated region of the genome, and the nucleocapsid protein gene of TCoV Histo pathological and direct immunohistochemical examinations were performed to detect TCoV antigen in infected intestine and bursa slides. All the results from stained tissues revealed lesions as described previously for TCoV infection. The direct immunohistochemical positive signal was present in all intestine slides. However, all bursa of Fabricius tissues analysed were negative. RT-PCR findings were positive for TCoV in all faecal droppings samples, and in 27% of cloacal swabs. Finally, the best field material for TCoV diagnosis was faecal droppings and/or intestine suspensions.

Metabolismo oxidativo de heterófilos de avestruzes jovens

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

Visual detection of turkey coronavirus RNA in tissues and feces by reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with hydroxynaphthol blue dye

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

Cryptosporidium infection in Brazil: implications for veterinary medicine and public health

O objetivo deste trabalho foi relatar, por meio de revisão de literatura, os resultados de pesquisas sobre a criptosporidiose no Brasil, com ênfase em sua ocorrência em animais e suas implicações em medicina veterinária e em saúde pública. Um número crescente de trabalhos sobre a infecção por Cryptosporidium spp. no Brasil está disponível na literatura nacional e internacional. Nestes trabalhos, são abordados principalmente aspectos relacionados à ocorrência de Cryptosporidium spp. em alimentos, amostras ambientais, no homem e em diversas espécies animais, particularmente em aves, bovinos, cães e gatos. Por meio de técnicas de biologia molecular, a maioria das espécies e alguns genótipos identificados em outros países foram descritos no Brasil. em mamíferos, houve identificação de C. bovis, C. canis, C. felis, C. meleagridis, C. parvum e o genótipo cervídeo; em diversas espécies de aves, foi descrita infecção por C. baileyi, C. galli, C. meleagridis, C. parvum e pelos genótipos I, II e III de aves. Várias espécies foram descritas no homem, como C. parvum e C. hominis, além de algumas espécies adaptadas a hospedeiros animais, como C. canis, C. felis e C. meleagridis.

Molecular characterization of Cryptosporidium spp. from fecal samples of birds kept in captivity in Brazil

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

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.

Aves como potenciais dispersoras de sementes de Ocotea pulchella Mart. (Lauraceae) numa área de vegetação de cerrado do sudeste brasileiro

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

Frugivoria e especificidade por hospedeiros na erva-de-passarinho Phoradendron rubrum (L.) Griseb. (Viscaceae)

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

Effects of frugivore impoverishment and seed predators on the recruitment of a keystone palm

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

Why are fruits colorful? The relative importance of achromatic and chromatic contrasts for detection by birds

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

Modelling post-release survival of reintroduced Red-billed Curassows Crax blumenbachii

Modelling post-release survival probabilities of reintroduced birds can help inform 'soft-release' strategies for avian reintroductions that use captive-bred individuals. We used post-release radiotelemetry data to estimate the survival probabilities of reintroduced captive-bred Red-billed Curassow Crax blumenbachii, a globally threatened Cracid endemic to the Brazilian Atlantic Rainforest. Between August 2006 and December 2008, 46 radiotagged Curassows from the Crax Brazil breeding centre were reintroduced to the Guapiacu Ecological Reserve (REGUA), Rio de Janeiro state, Brazil, in seven different cohorts. Reintroduced birds were most vulnerable during the first 12 months post-release from natural predation, domestic dogs and hunting. Annual post-release survival probability was high (75%) compared with published estimates for other Galliform species. However, when considering survival in all birds transported to REGUA (some birds died before release or were retained in captivity) and not only post-release survival, phi in this study was closer to estimates for other species (60%). The duration of the pre-release acclimatization period within the soft-release enclosure and the size of the released cohorts both positively influenced post-release survival of reintroduced Curassows. Our results are relevant to future Cracid reintroductions and highlight the importance of utilizing post-release monitoring data for evidence-based improvements to soft-release strategies that can significantly enhance the post-release survival of captive-bred birds.

Metabolic, hygric and ventilatory physiology of a hypermetabolic marsupial, the honey possum (Tarsipes rostratus)

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