Anna Paula de Oliveira

Influence of iron on mineral status of two rice (Oryza sativa L.) cultivars

Received: 02 April 2007; Returned for revision: 02 July 2007; Accepted: 14 August 2007Iron is an essential nutrient for plants. In aerobic conditions, Fe is highly unavailable for plant uptake, and Fe deficiencycan be severe in plants grown in calcareous soils. In waterlogged soils, however, Fe availability increases and can reachtoxic concentrations. Rice is an important staple crop worldwide and faces iron deficiency or excess, depending on thegrowth conditions. To contribute to the study of mechanisms involved in response to Fe deficiency and resistance to Feexcess, experiments were carried out with rice cultivars BR-IRGA 409 (I409, susceptible to Fe toxicity) and EPAGRI 108(E108, resistant to Fe toxicity) grown in culture solutions and submitted to Fe excess, control concentration ordeficiency (500, 6.5 or zero mg L

Nanobacteria-like particles: a threat to cell cultures

The main goal of this study is to alert researchers who work with cell cultures for the risk of contamination by structures called nanobacteria (NB). NB are tiny structures with size varying from 80 to 500 nm, commonly occurring in clusters and producing a biofilm which contains carbonate or hydroxyl apatite. The most likely source of cell culture contamination by such organisms is serum used as supplement in culture media. The presence of NB leads to a progressive culture deterioration with accumulation of granules (probably phagocytized NB) in cytoplasmic vacuoles, an increasing number of dead cells in the supernatant and degeneration of cells that remained attached to the bottom of the vessel. NB can also be found in culture supernatants where they are found in clusters with variable size and displaying brownian movement. In this study, 19 cell lineages, 8 batches of sera and 1 batch of growth supplement from different sources were analyzed. Samples from sera were cultured in Eagle’s Minimum Essential Medium (E-MEM) or incubated directly at 37oC. Tests carried out to detect the presence of extracellular bacteria, Mycoplasma sp and viruses were all negative. Analysis by scanning electron microscopy (SEM) revealed tiny oval structures less than 500 nm in size, isolated or in small groups, in all material analyzed except in one fetal bovine serum batch.

Construction and characterization of a bovine herpesvirus 5 mutant with a deletion of the gI, gE and US9 genes

Bovine herpesvirus 5 (BoHV-5) is a important cause of viral encephalitis in cattle in South America. Within the framework of developing a differential vaccine against BoHV-5, a deletion mutant was constructed based on a Brazilian BoHV-5 isolate. The target of the deletions were genes that code proteins implicated in the neurovirulence of BoHV-5, the glycoprotein I (gI), glycoprotein E (gE) and membrane protein US9. To construct the deletion mutant of BoHV-5, the flanking regions of all three genes were cloned in a prokaryotic plasmid. This deletion fragment was co-transfected with the viral DNA into bovine cells. Identification of deletion mutants was performed by immunostaining with an anti-gE monoclonal antibody. One of the gE negative viral populations found was purified, amplified and further examined by restriction endonuclesase analysis of its genomic DNA. The plaque sizes and penetration kinetics of the deletion mutant and wild type viruses were compared. The plaque sizes of the deletion mutant were significantly smaller than those of the parental strain (p ≤ 0.05), but no statistical differences were observed in penetration kinetics. The results indicate that the gI/ gE/US9 deletion mutant of BoHV-5 may have a reduced virulence in the host and is still viable enough to be a good candidate for the development of a BoHV-5 vaccine.

Caracterização antigênica e molecular de oito amostras do vírus da doença de Aujeszky isoladas no estado do Rio Grande do Sul em 2003

Pseudorabies or Aujeszkys disease (AD), caused by pseudorabies virus (PRV) is a major concern in swine production. In the state of Rio Grande do Sul, Brazil, AD was only detected in 1954, in cattle. In 2003 two outbreaks of encephalitis occurred on the northern region of the state, close to the border with the state of Santa Catarina. Pseudorabies virus (PRV) was isolated from distinct farms within the region and subjected to antigenic and genomic analyses. These isolates were compared with prototype strains NIA-3 and NP. Antigenic characterization with a panel of monoclonal antibodies (Mabs) directed to viral glycoproteins (gB, gC, gD and gE,) was performed by an imunoperoxidase monolayer assay (IPMA) on infected cell monolayers. Genomic characterization was carried out by restriction enzyme analysis (REA) of the whole DNA viral genome with Bam HI. The antigenic profile of the eight isolates from Rio Grande do Sul as well as strains NIA-3 and NP were similar. REA analysis revealed that all isolates from Rio Grande do Sul displayed a genomic type II arrangement, a genotype often found in other outbreaks of AD previously reported in other Brazilian states. The results obtained suggest that the eight isolates examined here were similar.

Field evaluation of safety during gestation and horizontal spread of a recombinant differential bovine herpesvirus 1 (BoHV-1) vaccine

Infeccoes pelo herpesvirus bovino tipo 1 (BoHV-1) sao importantes causas de doenca respiratoria, reprodutiva e abortos em bovinos. A vacinacao e frequentemente empregada para minimizar as perdas produzidas pela infeccao. Todavia, a imunizacao de vacas durante a prenhez com algumas vacinas contendo virus vivo modificado (MLV) pode ocasionalmente causar abortos. Em trabalho previo, nosso grupo desenvolveu uma vacina recombinante de BoHV-1 construida a partir de um isolado brasileiro de BoHV-1 (Franco et al., 2002a) do qual o gene que codifica para a glicoproteina E (gE) foi artificialmente deletado. Tal recombinante (gE-) vem sendo avaliado como vacina diferencial, isto e, capaz de permitir a diferenciacao entre animais vacinados e infectados. No presente estudo, o potencial de disseminacao do virus recombinante foi avaliado em um rebanho de gado de corte, em condicoes de campo. Para tanto, a seguranca da vacina gE- quando aplicada durante a prenhez foi avaliada pela inoculacao intramuscular de 107,4 doses infectantes para 50% dos cultivos celulares (DICC50) do virus em 22 femeas prenhes (14 previamente soronegativas e 8 previamente soropositivas para BoHV-1) em diferentes fases da gestacao. Outras 15 vacas prenhes foram mantidas como controles nao-vacinados. Nao ocorreram abortos, natimortos ou anormalidades fetais em nenhum dos grupos. Soroconversao foi observada nas femeas vacinadas previamente soronegativas. Em um segundo experimento, 4 novilhas foram inoculadas pela via intranasal com 107,6 DICC50 do virus recombinante, sendo mantidos em contato com 16 novilhas em uma area de campo, a uma densidade de 1 animal por hectare. Os animais foram monitorados quanto a presenca de sinais clinicos; amostras de soro foram coletadas nos dias 0, 30, 60 e 180 apos a vacinacao. Soroconversao foi observada apenas nos animais vacinados e nao nos contatos. Estes resultados indicam que, nas condicoes do presente estudo, a vacina gE- nao tem efeitos deleterios para femeas gestantes nem para seus fetos e nao se dissemina horizontalmente no rebanho.

Efficacy of a gE-deleted, bovine herpesvirus 1 (BoHV-1) inactivated vaccine.

Bovine herpesvirus type 1 (BoHV-1) is recognized as a major cause of economic losses in cattle. Vaccination has been widely applied to minimize losses induced by BoHV-1 infections. We have previously reported the development of a differential BoHV-1 vaccine, based on a recombinant glycoprotein E (gE)-deleted virus (265gE-). In present paper the efficacy of such recombinant was evaluated as an inactivated vaccine. Five BoHV-1 seronegative calves were vaccinated intramuscularly on day 0 and boostered 30 days later with an inactivated, oil adjuvanted vaccine containing an antigenic mass equivalent to 107.0 fifty per cent cell culture infectious doses (CCID50) of 265gE-. Three calves were kept as non vaccinated controls. On day 60 post vaccination both vaccinated and controls were challenged with the virulent parental strain. No clinical signs or adverse effects were seen after or during vaccination. After challenge, 2/5 vaccinated calves showed mild clinical signs of infection, whereas all non vaccinated controls displayed intense rhinotracheitis and shed virus for longer and to higher titres than vaccinated calves. Serological responses were detected in all vaccinated animals after the second dose of vaccine, but not on control calves. Following corticosteroid administration in attempting to induce reactivation of the latent infection, no clinical signs were observed in vaccinated calves, whereas non vaccinated controls showed clinical signs of respiratory disease. In view of its immunogenicity and protective effect upon challenge with a virulent BoHV-1, the oil adjuvanted preparation with the inactivated 265gE- recombinant was shown to be suitable for use as a vaccine.