IDENTIFICATION OF CANINE VISCERAL LEISHMANIASIS IN A PREVIOUSLY UNAFFECTED AREA BY CONVENTIONAL DIAGNOSTIC TECHNIQUES AND CELL-BLOCK FIXATION

After the report of a second case of canine visceral leishmaniasis (CVL) in São Bento da Lagoa, Itaipuaçu, in the municipality of Maricá, Rio de Janeiro State, an epidemiological survey was carried out, through active search, totaling 145 dogs. Indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and rapid chromatographic immunoassay based on dual-path platform (DPP(r)) were used to perform the serological examinations. The parasitological diagnosis of cutaneous fragments was performed by parasitological culture, histopathology, and immunohistochemistry. In the serological assessment, 21 dogs were seropositive by IFA, 17 by ELISA, and 11 by DPP(r), with sensitivity of 66.7%, 66.7% and 50%, and specificity of 87.2%, 90.2% and 94%, respectively for each technique. The immunohistochemistry of bone marrow using the cell-block technique presented the best results, with six positive dogs found, three of which tested negative by the other parasitological techniques. Leishmania sp. was isolated by parasitological culture in three dogs. The detection of autochthonous Leishmania infantum in Itaipuaçu, and the high prevalence of seropositive dogs confirm the circulation of this parasite in the study area and alert for the risk of expansion in the State of Rio de Janeiro.

Longitudinal study of the indirect immunofluorescence and complement fixation tests for diagnosis of chagas' disease in immunosuppressed patients submitted to renal transplantation

Clinical and serological follow-up of 7 patients submitted to renal transplantation and presenting positive serological reactions to Chagas 'disease before immunossupression did not show significant changes in indirect immunofluorescence and complement fixation titres for Chagas ' disease, or signs and symptoms indicating exacerbation of the disease during follow- up. In addition, 18 of 66 recipients of renal transplants considered to be non-chagasic before immunosuppression showed at least one positive result to the indirect immunofluorescence test for Chagas ' disease during the study period. The results suggest that the immunosuppression State induced in chagasic patients submitted to renal transplant did notpromoted exacerbation of the chronic infection in these patients and not interfere with the serological response of chronic chagasics, thus permitting the use of these serologic reactions for diagnostic purposes in these cases. However, the positive results ofthe indirect immunofluorescence test in non- chagasic patients indicate the needforjudicious interpretation ofthe indirect immunofluorescence test for the diagnosis of Chagas' disease in renal transplanted patients.

Process of fixation, imobilization and mineralization of ammonium in soil using N-15

The organic and inorganic forms of soil nitrogen and how they participate in the process of fixation, immobilization and mineralization of ammonium in soils were evaluated, after different periods of incubaton, utilizing two soils, a Lithic Haplustoll and a Typic Eutrorthox. The results obtained permit to suggest that : 1) The method for determination of the ammonium fixing capacity based on the extraction with 2N KC1, is considered to be subject to interferences of other soil fractions capable of retaining ammonium. 2) The increase in exchangeable ammonium content is related to the decrease in amino acids and hydrolyzable ammonium. 3) The immobilization and mineralization processes are still held under mil microbial. The forms more affected by this condition are amino acids and hydrolyzable ammonium.

Factors affecting phosphate fixation in soils of the state of São Paulo

A laboratory experiment was carried out to study the effects of chemical and physical characteristics of the soil on the phosphate fixing capacity. One hundred samples collected from various localities were at first characterized chemically and their particlesize distribution determined. They were then tested as to their phosphate fixing capacities. The results obtained were statistically analysed by means of both simple linear and multiple correlation. The following conclusions could be drawn: 1. simple linear regression analysis indicated that % C, exchangeable Al+3, CEC, % clay, pH and % sand were the soil characteristics which significantly affected phosphate fixing capacity of São Paulo State soils; 2. multiple linear regression analysis indicated that % C, exchangeable Mg(+2)9 exchangeable- Al+3 and % clay were the soil characteristics which significantly affected the phosphate fixing capacity of São Paulo State soils; 3. the phosphate phonomena fixing as they occur in the soils of the São Paulo State can be best described by the following equation: Y = -2,266 - 3,484 + 3,514 + 5,559 + 1,005 %C Mg+2 Al+3 % clay exchangeable exchangeable 4. phosphate fixation in the soil is affected by the combined effects of both soil chemical and physical characteristics.

Seeds enriched with phosphorus and molybdenum improve the contribution of biological nitrogen fixation to common bean as estimated by 15n isotope dilution

Seeds with a high concentration of P or Mo can improve the growth and N accumulation of the common bean (Phaseolus vulgaris L.), but the effect of enriched seeds on biological N2 fixation has not been established yet. This study aimed to evaluate the effect of seeds enriched with P and Mo on growth and biological N2 fixation of the common bean by the 15N isotope dilution technique. An experiment was carried out in pots in a 2 x 3 x 2 x 2 factorial design in randomized blocks with four replications, comprising two levels of soil applied P (0 and 80 mg kg-1), three N sources (without N, inoculated with rhizobia, and mineral N), two seed P concentrations (low and high), and two seed Mo concentrations (low and high). Non-nodulating bean and sorghum were used as non-fixing crops. The substrate was 5.0 kg of a Red Latosol (Oxisol) previously enriched with 15N and mixed with 5.0 kg of sand. Plants were harvested 41 days after emergence. Seeds with high P concentration increased the growth and N in shoots, particularly in inoculated plants at lower applied P levels. Inoculated plants raised from high P seeds showed improved nodulation at both soil P levels. Higher soil P levels increased the percentage of N derived from the atmosphere (%Ndfa) in bean leaves. Inoculation with the selected strains increased the %Ndfa. High seed P increased the %Ndfa in inoculated plants at lower soil P levels. High seed Mo increased the %Ndfa at lower soil P levels in plants that did not receive inoculation or mineral N. It is concluded that high seed P concentration increases the growth, N accumulation and the contribution of the biological N2 fixation in the common bean, particularly in inoculated plants grown at lower soil P availability.

Growth, nodulation and nitrogen fixation of cowpea in soils amended with composted tannery sludge

Tannery wastes generation is increasing every year and a suitable method for tannery sludge management is necessary in order to decrease this environmental problem. The composting is recognized as a suitable method for sludge recycling.. The effect of tannery sludge compost (TSC) rates on growth, nodulation and N fixation of cowpea was investigated. Sandy and clayey soils were amended with TSC at rates of 0, 7.5, 15, 30, and 60 t ha-1. The shoot dry weight of cowpea plants 45 days after emergence (DAE) was greater in the TSC-amended than in the unamended soil. In the sandy soil, nodule dry weight increased with TSC application 45 DAE. In the clayey soil, 45 DAE, nodule dry weight decreased with TSC amendment levels greater than 7.5 t ha-1 compared to the unamended control. The application of TSC increased N accumulation in the cowpea plants. The results suggest that cowpea responds differently to TSC depending on the amendment rate and initial soil type.

Growth and yield of common bean cultivars at two soil phosphorus levels under biological nitrogen fixation

The genotypic differences on growth and yield of common bean (Phaseolus vulgaris L.) in response to P supply were evaluated in a field experiment under biological N2 fixation. Eight cultivars were grown at two levels of applied P (12 and 50 kg ha-1 of P -- P1 and P2 respectively), in randomized block design in factorial arrangement. Vegetative biomass was sampled at three ontogenetic stages. The effects of genotype and phosphorus were significant for most traits, but not the genotype ´ phosphorus interaction. The cultivars presented different patterns of biomass production and nutrient accumulation, particularly on root system. At P1, P accumulation persisted after the beginning of pod filling, and P translocation from roots to shoots was lower. The nodule senescence observed after flowering might have reduced N2 fixation during pod filling. The responses of vegetative growth to the higher P supply did not reflect with the same magnitude on yield, which increased only 6% at P2; hence the harvest index was lower at P2. The cultivars with highest yields also presented lower grain P concentrations. A sub-optimal supply of N could have limited the expression of the yield potential of cultivars, reducing the genotypic variability of responses to P levels.

Effect of water stress on nitrogen fixation and nodule structure of common bean

The aim of this work was to investigate the effect of water stress on N2 fixation and nodule structure of two common bean (Phaseolus vulgaris L.) cultivars Carioca and EMGOPA-201. Plants were harvested after five and eight days of water stress. Carioca had lower nodule dry weight on both water stress periods; shoot dry weight was lower at five days water stress and did not differ from control after eight days stress. Both cultivars had lower nitrogenase activity than control after five and eight days water stress. For both cultivars, after eight days stress bacteroid membranes were damaged. Carioca presented more pronounced damage to infected tissue, with host cell vacuolation and loss of the peribacteroid membrane at five days after stress; at eight days after stress, there was degradation of cytoplasm host cells and senescence of bacteroids, with their release into intercellular spaces. Intensity of immunogold-labeling of intercellular cortical glycoprotein with the monoclonal antibodies MAC 236/265 was different for both cultivars.

Nitrogen fixation by groundnut and velvet bean and residual benefit to a subsequent maize crop

Chemical fertilisers are rarely avaiable to poor farmers, for whom the nitrogen (N) is often the most limiting element for cereal grain production. The objective of this study was to quantify the contribution of biological nitrogen fixation (BNF) to groundnut (Arachis hypogaea) and velvet bean (Mucuna pruriens) crops using the 15N natural abundance (delta15N) technique and to determine their residual effect and that of a natural fallow, on growth and N accumulation by two rustic maize varieties. The contribution of BNF calculated from delta15N data was 40.9, 59.6 and 30.9 kg ha-1, for groundnut, velvet bean and the natural fallow, respectively. The only legume grain harvested was from the groundnut, which yielded approximately 1.000 kg ha-1. The subsequent maize varieties ("Sol de Manhã" and "Caiana Sobralha") yielded between 1.958 and 2.971 kg ha-1, and were higher after velvet bean for both maize varieties and "Sol da Manhã" groundnut, followed by "Caiana" after groundnut and, finally, the natural fallow. For a small-holder producer the most attractive system is the groundnut followed by maize, as, in this treatment, both groundnut and maize grain harvest are possible. However, a simple N balance calculation indicated that the groundnut-maize sequence would, in the long term, deplete soil N reserves, while the velvet bean-maize sequence would lead to a build up of soil nitrogen.

Genetic characterization and nitrogen fixation capacity of Rhizobium strains on common bean

This study aimed to genetically characterize four new Rhizobium strains, and to evaluate their nodulation and fixation capacity compared to commercial strains and to native rhizobia population of a Brazilian Rhodic Hapludox. Two experiments were carried out in randomized blocks design, under greenhouse conditions, in 2007. In the first experiment, the nodulation and nitrogen fixation capacity of new strains were evaluated, in comparison to the commercial strains CIAT-899 and PRF-81 and to native soil population. It was carried out in plastic tubes filled with vermiculite. DNA extractions and PCR sequencing of the intergenic space were made from the isolated pure colonies, in order to genetically characterize the strains and the native rhizobia population. In the second experiment, the nodulation and productivity of common beans Perola cultivar were determined, with the use of evaluated strains, alone or in mixture with PRF-81 strain. It was carried out in pots filled with soil. The native soil population was identified as Rhizobium sp. and was inefficient in nitrogen fixation. Three different Rhizobium species were found among the four new strains. The LBMP-4BR and LBMP-12BR new strains are among the ones with greatest nodulation and fixation capacity and exhibit differential responses when mixed to PRF-81.

Diversity and nitrogen fixation efficiency of rhizobia isolated from nodules of Centrolobium paraense

The objective of this work was to isolate and characterize rhizobia from nodules of Centrolobium paraense and to evaluate their symbiotic efficiency. Soil samples collected from four sites of the Roraima Cerrado, Brazil, were used to cultivate C. paraense in order to obtain nodules. Isolates (178) were obtained from 334 nodules after cultivation on medium 79. Twenty-five isolates belonging to six morphological groups were authenticated using Vigna unguiculata and they were characterized by 16S rRNA. Isolates identified as Bradyrhizobium were further characterized using rpoB gene sequencing. A greenhouse experiment was carried out with C. paraense to test the 18 authenticated isolates. Approximately 90% of the isolates grew slowly in medium 79. The 16S rRNA analysis showed that 14 authenticated isolates belong to the genus Bradyrhizobium, and rpoB indicated they constitute different groups compared to previously described species. Only four of the 11 fast-growing isolates nodulated V. unguiculata, two of which belong to Rhizobium, and two to Pleomorphomonas, which was not previously reported as a nodulating genus. The Bradyrhizobium isolates ERR 326, ERR 399, and ERR 435 had the highest symbiotic efficiency on C. paraense and showed a contribution similar to the nitrogen treatment. Centrolobium paraense is able to nodulate with different rhizobium species, some of which have not yet been described.

Changes in N2 fixation in Stylosanthes scabra derived from tissue culture

Plants were regenerated from leaf-derived callus culture of Stylosanthes scabra, a polyploid legume tolerant to drought and adapted to acid soils. A total of 168 regenerants were planted out in Leonard jars in a complete randomized design. Nitrogen fixation and vegetative growth were indirectly evaluated by shoot dry weight, root dry weight, shoot N content and acetylene reduction activity. The results showed higher variation in the regenerants than in controls not submitted to tissue culture. Significant differences were found for all nitrogen fixation related-traits

Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense

NifA protein activates transcription of nitrogen fixation operons by the alternative sigma54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS) located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST)-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.