Serologic and molecular diagnostic and bioassay in mice for detection of Toxoplasma gondii in free ranges chickens from Pantanal of Mato Grosso do Sul

The aim of this study was to investigate the occurrence of Toxoplasma gondii and compare the results obtained in the Modified Agglutination Test (MAT), Polimerase Chain Reaction (PCR) and bioassay in mice. In order to accomplish this, 40 free-range chickens from eight farms in neighboring areas to the Pantanal in Nhecolândia, Mato Grosso do Sul, were euthanized and blood samples, brain and heart were collected. The occurrence of anti-T. gondii antibodies found in chickens was 67.5% (27 samples), considering as a cutoff point the dilution 1:5. Among the samples analyzed, 7 (25.9%) were positive in the dilution 1:5, 3 (11.1%) in 1:10, 2 (7.4%) in 1:20, 3 (11.1%) in 1:320, 1 ( 3.7%) in 1:640, 3 (11.1%) in 1:1280, 2 (7.4%) in 1:2560, 4 (14.8%) in 1:5120 and 2 (7.4%) in 1:10.240. From the mixture of tissue samples (brain and heart) from the chickens analyzed, 16 (40%) presented electrophoretic bands compatible with T. gondii by PCR (gene B1). In the comparison of techniques, 59.26% positivity in PCR was revealed among animals that were seropositive in MAT (cutoff 1:5). From 141 inoculated mice, six (4.44%) died of acute toxoplasmosis between 15 and 23 days after inoculation. Surviving mice were sacrificed at 74 days after inoculation, and a total of 28 cysts were found in the brains of 10 distinct groups. From the seropositive hens, 27 bioassays were performed and 11 (40.7%) isolates were obtained. A greater number of isolations happened in mice that were inoculated with tissues from chickens that had high titers for anti-T. gondii antibodies. Chronic infection in mice was observed in nine groups (33.3%) from five different properties. Among the surviving mice, 25.6% were positive for T. gondii in MAT (1:25). From mice positive in PCR, 87.5% were also positive in MAT. Among the PCR-negative mice, 5.2% were positive for T. gondii in MAT. It can be concluded through this study that the occurrence of infecton by T. gondii in the rural properties studied was high, that PCR directed to gene B1 does not confirm the viability of the parasite, but it can be used as a screening method for the selection of chickens infected by T. gondii, that the animals with titer greater than 10 must be prioritized for the selection of animals for bioassay, since for them, the chances of isolating the parasite are greater and that seroconversion in experimentally infected mice is not a good indicator for isolating the agent.

Leishmanicidal and cholinesterase inhibiting activities of phenolic compounds of Dimorphandra gardneriana and Platymiscium floribundum, native plants from Caatinga biome

In recent years, the Brazilian Health Ministry and the World Health Organization have supported research into new technologies that may contribute to the surveillance, new treatments, and control of visceral leishmaniasis within the country. In light of this, the aim of this study was to isolate compounds from plants of the Caatinga biome, and to investigate their toxicity against promastigote and amastigote forms of Leishmania infantum chagasi, the main responsible parasite for South American visceral leishmaniasis, and evaluate their ability to inhibit acetylcholinesterase enzyme (AChE). A screen assay using luciferase-expressing promastigote form and an in situ ELISA assay were used to measure the viability of promastigote and amastigote forms, respectively, after exposure to these substances. The MTT colorimetric assay was performed to determine the toxicity of these compounds in murine monocytic RAW 264.7 cell line. All compounds were tested in vitro for their anti-cholinesterase properties. A coumarin, scoparone, was isolated from Platymiscium floribundum stems, and the flavonoids rutin and quercetin were isolated from Dimorphandra gardneriana beans. These compounds were purified using silica gel column chromatography, eluted with organic solvents in mixtures of increasing polarity, and identified by spectral analysis. In the leishmanicidal assays, the compounds showed dose-dependent efficacy against the extracellular promastigote forms, with an EC50 for scoporone of 21.4µg/mL, quercetin and rutin 26 and 30.3µg/mL, respectively. The flavonoids presented comparable results to the positive control drug, amphotericin B, against the amastigote forms with EC50 for quercetin and rutin of 10.6 and 43.3µg/mL, respectively. All compounds inhibited AChE with inhibition zones varying from 0.8 to 0.6, indicating a possible mechanism of action for leishmacicidal activity.

Mapping the sites of latency and reactivation by bovine herpesvirus 5 (BoHV-5) and a thymidine kinase-deleted BoHV-5 in lambs

A thymidine kinase (tk)-deleted bovine herpesvirus 5 (BoHV-5tkΔ) was previously shown to establish latent infection and reactivate - even poorly - in a sheep model (Cadore et al. 2013). As TK-negative alphaherpesviruses are unlike to reactivate in neural tissue, this study investigated the sites of latency and reactivation by this recombinant in lambs. For this, groups of lambs were inoculated intranasally with the parental BoHV-5 strain (SV-507/99) or with the recombinant BoHV-5tkΔ. During latent infection (40 days post-inoculation, pi), the distribution of recombinant virus DNA in neural and non-neural tissues was similar to that of the parental virus. Parental and recombinant virus DNA was consistently detected by PCR in trigeminal ganglia (TGs); frequently in palatine and pharyngeal tonsils and, less frequently in the retropharyngeal lymph nodes. In addition, latent DNA of both viruses was detected in several areas of the brain. After dexamethasone (Dx) administration (day 40pi), the recombinant virus was barely detected in nasal secretions contrasting with marked shedding of the parental virus. In tissues of lambs euthanized at day 3 post-Dx treatment (pDx), reverse-transcription-PCR (RT-PCR) for a late viral mRNA (glycoprotein D gene) demonstrated reactivation of parental virus in neural (TGs) and lymphoid tissues (tonsils, lymph node). In contrast, recombinant virus mRNA was detected only in lymphoid tissues. These results demonstrate that BoHV-5 and the recombinant BoHV-5tkΔ do establish latent infection in neural and non-neural sites. Reactivation of the recombinant BoHV-5tkΔ, however, appeared to occur only in non-neural sites. In anyway, the ability of a tk-deleted strain to reactivate latent infection deserves attention in the context of vaccine safety.

Spontaneous poisoning by Ricinus communis (Euphorbiaceae) in cattle

The aim of this study is to report cases of spontaneous poisoning of cattle by Ricinus communis (castor beans) in Paraíba, a semiarid region of northeastern Brazil. The cases were observed in 2 herds on neighboring properties in 2013. Clinical signs developed within 6-24 h and consisted of weakness, tachycardia, dyspnea, profuse watery diarrhea, dehydration, depression, instability, cramps, permanent lateral recumbency and death within 48-72 h. Of the 60 cattle at risk, 19 were affected and 14 died. Five fully recovered after the course of 12 days. Three animals were necropsied. The main gross lesions were hemopericardium, hemothorax, pulmonary edema, petechial hemorrhages in the epicardium and endocardium, ecchymoses at the papillary muscles and suffusions on the intercostal muscles. Hemorrhages were also observed in the abdominal cavity, spleen and mucosa of the abomasum and small intestine. The rumen content was liquid with a large amount of castor bean seeds. There were circular, whitish and focally diffuse areas in the liver parenchyma. The main microscopic lesions consisted of multifocal coagulative myocardial necrosis with the presence of mononuclear cell infiltration and varying degrees of bleeding between cardiac muscle fibers. The abomasum and small intestine mucosae and submucosa had mild edema and mononuclear and polymorphonuclear inflammatory cell infiltration. The diagnosis of R. communis was based on the history of plant consumption, clinical signs, pathology of the disease and the presence of large amounts of castor bean seeds in the forestomachs.

Pasteurella multocida type A as the primary agent of pneumonia and septicaemia in pigs

Abstract: In order to understand better the pathological aspects and spread of Pasteurella multocida type A as the primary cause of pneumonia in pigs, was made an experiment with intranasal inoculation of different concentrations of inocula [Group (G1): 108 Colony Forming Units (CFU)/ml; G2: 107 CFU/ml; G3: 106 CFU/ml and G4: 105 CFU/ml], using two pigs per group. The pigs were obtained from a high health status herd. Pigs were monitored clinically for 4 days and subsequently necropsied. All pigs had clinical signs and lesions associated with respiratory disease. Dyspnoea and hyperthermia were the main clinical signs observed. Suppurative cranioventral bronchopneumonia, in some cases associated with necrosuppurative pleuropneumonia, fibrinous pericarditis and pleuritic, were the most frequent types of lesion found. The disease evolved with septicaemia, characterized by septic infarctions in the liver and spleen, with the detection of P. multocida type A. In this study, P. multocida type A strain #11246 was the primary agent of fibrinous pleuritis and suppurative cranioventral bronchopneumonia, pericarditis and septicaemia in the pigs. All concentrations of inoculum used (105-108 CFU/ml) were able to produce clinical and pathological changes of pneumonia, pleuritis, pericarditis and septicemia in challenged animals.

Carryover effect of fomesafen, applied on edible bean, on sucessional maize

Carryove reffects of fomesafen on successional maize were studied in clay soil. Fomesafen was applied as postemergence at Five rate s (0; 0.12 5: 0.25 ; 0.37 5, and 0.5 kg/ha-1) to edible beans. Maize was planted 198 and 65 days after fomesafen application in 1992 and 212 and 65 days after fomesafen application in 1993. Fomesafen residues were detected in soils up to 20cm depth but residue concentration was higher in 0-10 cm soil depth. Fomesafen residues reduced leaf chlrophyll content and root volume of 10 days old maize when planted 65 days after application but were not affected when planted 212 days after application. However, the decreases in leaf chlorophyll and root volume did not affect the maize yield.

Weed control in young coffee plantations through post emergence herbicide application onto total area

This study was carried out to investigate the efficiency of several herbicides under field conditions, by post-emergence application onto the entire area, their effect on the control of weeds in young coffee plantations and commercial coffee and bean intercropping system, as well as on both crops. Seedlings of Coffea arabica cv. Red Catuaí with four to six leaf pairs were transplanted to the field and treated according to conventional agronomic practices. A bean and coffee intercropping system was established by sowing three lines of beans in the coffee inter-rows. At the time the herbicides were sprayed, the coffee plants had six to ten leaf pairs; the bean plants, three leaflets; and the weeds were at an early development stage. Fluazifop-p-butyl and clethodim were selective for coffee plants and controlled only Brachiaria plantaginea and Digitaria horizontalis efficiently. Broad-leaved weeds (Amaranthus retroflexus, Bidens pilosa, Coronopus didymus, Emilia sonchifolia, Galinsoga parviflora, Ipomoea grandifolia, Lepidium virginicum, and Raphanus raphanistrum) were controlled with high efficiency by sole applications of fomesafen, flazasulfuron, and oxyfluorfen, except B. pilosa, C. didymus, and R. raphanistrum for oxyfluorfen. Sequential applications in seven-day intervals of fomesafen + fluazifop-p-butyl, or clethodim, and two commercial mixtures of fomesafen + fluazifop-p-butyl simultaneously controlled both types of weed. Cyperus rotundus was only controlled by flazasulfuron. Except for fluazifop-p-butyl and clethodim, all herbicide treatments caused only slight injuries on younger coffee leaves. However, further plant growth was not impaired and coffee plant height and stem diameter were therefore similar in the treatments, as evaluated four months later. Fomesafen, fluazifop-p-butyl, and clethodim, at sole or sequential application, and the commercial mixtures of fomesafen + fluazifop-p-butyl were also highly selective for bean crop; thus at doses recommended for bean crop, these herbicides may be applied to control weeds in coffee and bean intercropping systems by spraying the entire area.

Development of bean plants in soil contaminated with trifloxysulfuron-sodium after Stizolobium aterrimum and Canavalia ensiformis cultivation

Phytoremediation, the use of plants to decontaminate soils and water resources from organic pollutants such as herbicides, is economically and environmentally a promising technique applied in many areas, including agriculture. The objective of this work was to evaluate the development of bean plants cultivated in the field, in soil with different levels of trifloxysulfuron-sodium contamination, following cultivation of two green manure species, as well as to evaluate the possibility of recontamination of the area by such herbicide with the straw permanence on the soil. The experiment was carried out in Coimbra, MG, Brazil, on a sandy clayey Red - Yellow Argisol from March to November 2003. Four levels of soil contamination with trifloxysulfuron-sodium (0.00; 3.75; 7.50; and 15.00 g ha-1) were used as well as the following five types of cultivation prior to bean sowing in the area after herbicide application: black velvet beans (Stizolobium aterrimum) followed by removal of straw; S. aterrimum, followed by permanence of straw; jack bean (Canavalia ensiformis), followed by removal of straw; C. ensiformis followed by permanence of straw; and without prior cultivation, weed-free (weeded control). The leguminous plants were kept in the area for 65 days, cut close to the soil, and with its aerial part left or not on the surface of the experimental plot, depending on the treatment. Fifteen days after the species were cut, bean was sown in the area. At 45 days after emergence (DAE) of the bean plants, plant height and dry mass of the aerial part were evaluated. Grain productivity was determined during harvest. Height, dry matter of the aerial part and grain productivity of the bean plants, cultivated in an area previously contaminated with trifloxysulfuron-sodium at any of the levels tested, were higher with prior cultivation of S. aterrimum or C. ensiformis. At the lowest level of herbicide contamination, prior cultivation of C. ensiformis was found to be more efficient than that of S. aterrimum in mitigating the harmful effects of trifloxysulfuron-sodium on bean grain production. The permanence of the straw of the green manure species during the bean cycle did not harm the development of the plants or caused culture productivity losses, indicating that straw permanence in the area does not promote recontamination of the area.

Weed control via intercropping with gliricidia: II. corn crop

A reduction in herbicide use is one of modern agriculture's main interests and several alternatives are being investigated with this objective, including intercropping. Gliricídia (Gliricidia sepium) mulch has no allelopathic effect on corn or beans but significantly decreased the population of some weed species. The objective of this study was to evaluate green ear and grain yield in corn cultivars as a response to weed control achieved via intercropping with gliricidia. A completely randomized block design with five replicates and split-plots was used. Cultivars AG 1051, AG 2060, BRS 2020, and PL 6880 (assigned to plots) were submitted to the following treatments: no hoeing, hoeing (performed at 20 and 40 days after sowing the corn), and corn intercropped with gliricidia. Gliricidia was grown in a transplanting system to ensure uniform germination and fast establishment in the field. Seeding was made in 200-cell trays with one seed per cell (35 mL volume). The plants emerged two to three days after sowing and were transplanted to a permanent site two to three days after emergence. Corn was sown on the same day gliricidia was transplanted. Sixteen weed species occurred at different frequencies, with uneven distribution in the experimental area. Cultivars AG 1051 and AG 2060 were the best with reference to most characteristics employed to evaluate green corn yield. Cultivar AG 1051 provided the highest grain yield. The highest green ear yield and grain yield values were obtained with hoeing. However, the fact that intercropped plots showed intermediate yield between the values obtained for hoed and non-hoed plots indicates that gliricidia was beneficial to corn, and exerted a certain level of weed control.

Weeds infestation in corn intercropped with forages at different planting densities

Corn is planted in the Center West region of Brazil as a second crop, following soybeans or beans. Intercropping of Brachiaria species with corn as a second crop increases the mulching in the cropping system. This study aimed to evaluate the weeds infestation in soybeans following corn/forages intercrop, as a function of corn plant structure, forage species and density. Experiments were conducted in a completely randomized blocks design with four replications, in Ponta Porã and Dourados municipalities, Mato Grosso do Sul state, Brazil, in 2010/2011. Treatments consisted of three corn hybrids with distinct plant architectures intercropped with three forage species: Brachiaria ruziziensis, B. brizantha and B.decumbens, at five densities, and the resulting dry mass was maintained throughout the winter. During the following cropping season, forages were desiccated prior to planting soybeans, and the dry mass of weeds, dry mass of the mulching, soil coverage by weeds, and the broadleaf/grass weed species index (WPI) were determined 15 days after soybean emergence, submitted to an F-test, and analyzed either by regression or by multiple mean comparison, according to the nature of the data. When intercropping corn with species of Brachiaria, a reduction in the overall weeds infestation may always be expected; among the studied forage species, more problems with weeds may be anticipated in areas with a less competitive species, e.g. B.ruziziensis. Under the conditions of the trials, B.brizantha and B.decumbens were more capable of inhibiting the emergence of weed species in the winter.

Host suitability of weeds and forage species to root-knot nematode meloidogyne graminicola as a funcion of irrigation management

The irrigated rice production can be limited by various phytopathogenic agents, including root-knot nematodes (Meloidogyne spp.). Thus, the aim of this research was to check the host suitability of plant species most often found off-season and during rice cultivation, to root-knot nematode Meloidogyne graminicola, under two irrigation managements. Two experiments were conducted in a completely randomized design. In the first experiment seven plant species that occur in an area of rice cultivation, in fallow, off-season were evaluated. For the second experiment nine weed species infesting the irrigated rice culture were tested in rainfed and flooding conditions. The sixteen species, kept individually in pots with sterilized substrate, were inoculated with 5,000 eggs and second stage juveniles (J2) of nematode. BRS 410 IRGA rice plants inoculated with M.graminicola were used as control. Two months after inoculation, the root system of each plant was evaluated for number of galls and nematode reproduction factor. It was verified that the species of off-season of rice cultivation Sida rhombifolia, Raphanus raphanistrum, Spergula arvensis, Lotus corniculatus and Trifolium repens, and, during the cycle of rice cultivation, Aeschynomene denticulata, Leersia hexandra, are immune to nematode. The plant species off-season, Avena strigosa and Lolium multiflorum and of cultivation, Alternanthera philoxeroides, red rice, Echinochloa crusgalli, Cyperus difformis, Cyperus esculentus, Cyperus iria and Fimbristylis miliacea would behave as hosts of M.graminicola, mostly under rainfed conditions.

Glyphosate drift affects arbuscular mycorrhizal association in coffee

Mycorrhizal association promotes better survival and nutrition of colonized seedling on field, and consequently, increasing of productivity. However, the weed management can interfere on this association, due to incorrect use of glyphosate. This work has assessed the effects of glyphosate drift on the growth and nutrition of arabica coffee plants (Catuaí Vermelho - IAC 99) colonized with arbuscular mycorrhizal fungi (AMF). The experiment was conducted in 2 x 5 factorial scheme, and included inoculated and non-inoculated plants, and five glyphosate subdoses (0.0, 57.6, 115.2, 230.4, and 460.8 g ha-1 of glyphosate), in randomized blocks with five replication. The inoculation was carried during the greenhouse phase of seedlings production with a mixture of Rhizophagus clarus and Gigaspora margarita, and after to transplanting, when the plants had seven pairs of leaves, glyphosate subdoses were applied. The product caused intoxication in up to 60% of non-inoculated and 45% on inoculated plants, when the highest dose of 460.8 g a.e. ha-1 was applied. A negative effect was noted on the growth and phosphorus content of coffee plants, this effect increased depending on glyphosate subdose, but regardless of inoculation. Glyphosate drift reduces the growth and nutrition of plants colonized by species of AMF and native fungi, negatively affecting root colonization of plants treated.

APPLICATION HEIGHT IN HERBICIDES EFFICIENCY IN BEAN CROPS

ABSTRACTInadequate herbicide application can result in failures in weed control and/or poisoning of the crops, resulting in yield losses. In this research were assessed the effects of the sprayer nozzle boom height in the distribution of the spray solution for weed control, influencing intoxication of beans and crop yield. Experiments were conducted in laboratory and field conditions. In laboratory, the performance of flat spray tip TT 11002 was assessed at heights 0.20, 0.30, 0.40 and 0.50 meters with respect to the target surface. In the field the same heights were assessed in applications of herbicides fomesafen, fluazifop-P-butyl and fomesafen + fluazifop-P-butyl. There was an inverse relationship between the height of the spray boom and the coefficients of variation of the patterns. The mixture better efficiency in a tank of fluazifop-P-butyl + fomesafen was obtained with the height of 0.50 m from the target. This treatment resulted in better weed control, lower poisoning of the bean plants and better crop yield rates.

Germination of spores and growth of gametophytes and sporophytes of Rumohra adiantiformis (Forst.) Ching (Dryopteridaceae) after spore cryogenic storage

Rumohra adiantiformis (Forst.) Ching is a fern (Dryopteridaceae) used in floral arrangements. Spores sterilized in 15% (v/v) solution of commercial sodium hypochlorite for 10 minutes and unsterilized spores were plunged in liquid nitrogen and held for 15 minutes and for 90 days. After the cryogenic treatments, spores were taken out of liquid nitrogen and rapidly thawed out in a water bath or slowly at room temperature and were cultured in Mohr's mineral solution as modified by Dyer, kept at 25 ± 2 ºC and a 16-hours photoperiod. Statistical differences were not observed in the germination of unsterilized spores immersed or not immersed in liquid nitrogen, but when the spores were previously sterilized, a severe inhibition of germination was observed in cryopreserved spores. Faster mean germination time was observed for unsterilized spores cryopreserved in liquid nitrogen for 15 minutes. The germination of spores stored in liquid nitrogen for 90 days reached the maximum percentage after 12 days, while control spores reached their maximum percentage after 16 days. Levels of soluble sugars did not vary among treatments in gametophytes cultivated for 10 weeks after spore inoculation. The number of fronds and the length of the longest frond on sporophytes did not differ statistically among treatments. The relative growth rate of sporophytes grown from cryopreserved and control spores were not statistically different among treatments. Spores of R. adiantiformis immersed in liquid nitrogen for 15 minutes apparently produced phenotypically normal plants.

Effectiveness of plant growth promoting rhizobacteria in facilitating lead and nutrient uptake by little seed canary grass

We investigated the effectiveness of Nitroxin inoculation on lead (Pb) and nutrient uptakes by little seed canary grass. The factors tested included inoculation (or not) with Nitroxin and different soil concentrations of Pb (0, 200, 400 and 800mgPbkg-1 soil). Increasing soil concentrations of Pb decreased stem, leaf and root dry weights. Shoot phosphorus concentrations increased in parallel with increasing soil Pb concentrations. Nitroxin inoculation did not alter the phosphorus concentration of the roots. The Pb translocation factor was >1 in inoculated treatments in the Pb soil concentration range of 200 to 400mgkg-1; the translocation factor for 800mgPbkg‑1 with no inoculation of Nitroxin was, however, <1. Our results indicated that the Pb bioaccumulation factor for little seed canary grass was <1, indicating that it is a Pb excluding plant.