COMPARISON OF SIX COMMERCIALLY-AVAILABLE DNA POLYMERASES FOR DIRECT PCR

SUMMARYThe use of a “direct PCR” DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens.

CORAL SNAKE ANTIVENOM PRODUCED IN CHICKENS (Gallus domesticus)

The production of anti-snake venom from large mammal's blood has been found to be low-yielding and arduous, consequently, antivenom immunoglobulins for treatment are achieved regularly as polyvalent serum. We have standardized an undemanding technique for making purified immunoglobulin IgY antivenom consisting of polyclonal antibodies against coral snake venom in the egg yolk of immunized hens. We have adapted a reported process of antibody purification from egg yolks, and achieved 90% antibody purity. The customized technique consisted of the removal of lipids from distilled water-diluted egg yolks by a freeze–thaw sequence. The specific immunoglobulins were present in the egg yolk for up to 180 days postimmunization. Therefore, by means of small venom quantities, a significant amount of immunoglobulins were found in an adequately purified state (The obtained material contained about 90% pure IgY). The antigen binding of the immunoglobulins was detected by a double immunodiffusion test. Titers of antibodies in the yolk were estimated with a serum protection assay (Median effective dose = ED50) (ED50= 477 mg/kg). Given that breeding hens is economically feasible, egg gathering is noninvasive and the purification of IgY antibodies is quick and easy, chicken immunization is an excellent alternative for the production of polyclonal antibodies. To the best of our knowledge, this is the first coral snake antivenom prepared in birds.

Ultrastructural study on the morphological changes to male worms of Schistosoma mansoni after in vitro exposure to allicin

INTRODUCTION: Garlic has a wide range of actions, including antibacterial, antiviral, antifungal, antiprotozoal and anthelmintic actions. This antiparasitic activity has been attributed to allicin, which is the main constituent of garlic. The present study aimed to investigate the in vitro activity of allicin on the tegument of adult Schistosoma mansoni worms using scanning electron microscopy. METHODS: Swiss Webster mice were infected with S. mansoni cercariae (100 per mouse) and sacrificed 50 days later to acquire the adult worms. These worms were collected by perfusion and placed in RPMI medium 1,640 at 37°C before transferring to RPMI media containing 0 (control), 5, 10, 15 and 20mg/mL of allicin, where they were incubated for 2h. The worms were fixed in 2.5% glutaraldehyde solution, washed twice, post-fixed in osmium tetroxide, washed twice and then dehydrated with ascending grades of ethanol. The samples were air-dried, mounted on stubs, gold coated in an ion sputtering unit and viewed using a scanning electron microscope. RESULTS: A concentration of 5mg/mL caused wrinkling in the tegument; a concentration of 10mg/mL resulted in changes to tubercles and loss or modification of spines. With 15 and 20mg/mL increasing damage to the tegument could be seen, such as vesicle formation and the presence of ulcers. CONCLUSIONS: These findings demonstrate the effect of allicin on adult S. mansoni worms and indicate that most of the changes occur at concentrations greater than that normally indicated for treatment.

Seroepidemiological study of human cysticercosis with blood samples collected on filter paper, in Lages, State of Santa Catarina, Brazil, 2004-2005

INTRODUCTION: Human serofrequency of antibodies against Taenia solium antigens was determined and risk factors for cysticercosis transmission were identified. METHODS: Individuals (n=878) from periurban and rural locations of Lages, SC, were interviewed to gather demographic, sanitary and health information. Interviews and blood sample collections by finger prick on Whatman filter paper were performed from August 2004 to May 2005. Observation determined that 850 samples were suitable for analysis and were tested by ELISA using vesicular fluid of Taenia crassiceps heterologous antigen. To ensure the reliability of the results, 77 samples of the dried blood were matched with sera. The reactive samples were submitted to a serum confirmatory immunoblot (IB) test using purified Taenia crassiceps glycoproteins. RESULTS: The ELISA results for the dried blood and serum samples were statistically consistent. ELISA was positive in 186 (21.9%) out of 850 individuals. A group of 213 individuals were asked to collect vein blood for IB (186 with positive result in ELISA and 27 with inappropriate whole blood samples) and 130 attended the request. The IB was positive in 29 (3.4%) out of 850 individuals. A significant correlation (p = 0.0364) was determined among individuals who tested positive in the IB assay who practiced both pig rearing and kitchen gardening. CONCLUSIONS: ELISA with dried blood eluted from filter paper was suitable for cysticercosis population surveys. In Lages, human infection was associated with pig rearing and kitchen gardening. The prevalence index was compatible with other Latin American endemic areas.

Epidemiological profile of Trypanosoma cruzi-infected mothers and live birth conditions in the state of Minas Gerais, Brazil

INTRODUCTION: The aim of this study was to assess the epidemiological characteristics of Trypanosoma cruzi-infected mothers and the live birth conditions of neonates.METHODS:A serological survey with IgG-specific tests was conducted using dried blood samples from newborn infants in the State of Minas Gerais. T. cruzi infection was confirmed in mothers through positive serology in two different tests, and infected mothers were required to have their infants serologically tested after the age of 6 months. The birth conditions of the neonates were obtained from the System of Information on Live Births database.RESULTS:The study included 407 children born to T. cruzi-infected mothers and 407 children born to uninfected mothers. The average age of seropositive mothers was 32 years (CI95% 31.3-32.6), which was greater than the average age of seronegative mothers - 25 years (CI95% 24.8-25.2). The mothers' level of education was higher among uninfected mothers (41% had 8 or more years of education, versus 22% between the infected mothers). Vaginal delivery was more frequent among infected mothers. There was no evidence of inter-group differences with respect to the child's sex, gestational age, birth weight or Appearance, pulse, grimace, activity and respiration (APGAR) scores at 1 and 5 minutes.Conclusions:The level of education and the greater number of previous pregnancies and cases of vaginal delivery reflect the lower socioeconomical conditions of the infected mothers. In the absence of vertical transmission, neonates had similar health status irrespective of the infection status of their mothers.

Antibacterial, anti-swarming and anti-biofilm formation activities of Chamaemelum nobile against Pseudomonas aeruginosa

AbstractINTRODUCTION:Chamomile ( Chamaemelum nobile ) is widely used throughout the world, and has anti-inflammatory, deodorant, bacteriostatic, antimicrobial, carminative, sedative, antiseptic, anti-catarrhal, and spasmolytic properties. Because of the increasing incidence of drug-resistant bacteria, the development of natural antibacterial sources such as medical herbs for the treatment of infectious diseases is necessary. Extracts from different plant parts such as the leaves, flowers, fruit, and bark of Combretum albiflorum, Laurus nobilis , and Sonchus oleraceus were found to possess anti-quorum sensing (QS) activities. In this study, we evaluated the effect of C. nobile against Pseudomonas aeruginosa biofilm formationMETHODS:The P. aeruginosa samples were isolated from patients with different types of infection, including wound infection, septicemia, and urinary tract infection. The flowers of C. nobile were dried and the extract was removed using a rotary device and then dissolved in dimethyl sulfoxide at pH 7.4. The microdilution method was used to evaluate the minimum inhibitory concentration (MIC) of this extract on P. aeruginosa , and biofilm inhibition was assayed.RESULTS:Eighty percent of the isolated samples (16/20) could form a biofilm, and most of these were isolated from wound infections. The biofilm inhibitory concentration of the C. nobile extract was 6.25-25mg/ml, whereas the MIC was 12.5-50mg/ml.CONCLUSIONS:The anti-QS property of C. nobile may play an important role in its antibacterial activity, thus offering an additional strategy in the fight against bacterial infections. However, molecular investigation is required to explore the exact mechanisms of the antibacterial action and functions of this phytocompound.

Soil mineral N dynamics beneath mixtures of leaves from legume and fruit trees in Central Amazonian multi-strata agroforests

Long term applications of leguminous green mulch could increase mineralizable nitrogen (N) beneath cupuaçu trees produced on the infertile acidic Ultisols and Oxisols of the Amazon Basin. However, low quality standing cupuaçu litter could interfere with green mulch N release and soil N mineralization. This study compared mineral N, total N, and microbial biomass N beneath cupuaçu trees grown in two different agroforestry systems, north of Manaus, Brazil, following seven years of different green mulch application rates. To test for net interactions between green mulch and cupuaçu litter, dried gliricidia and inga leaves were mixed with senescent cupuaçu leaves, surface applied to an Oxisol soil, and incubated in a greenhouse for 162 days. Leaf decomposition, N release and soil N mineralization were periodically measured in the mixed species litter treatments and compared to single species applications. The effect of legume biomass and cupuaçu litter on soil mineral N was additive implying that recommendations for green mulch applications to cupuaçu trees can be based on N dynamics of individual green mulch species. Results demonstrated that residue quality, not quantity, was the dominant factor affecting the rate of N release from leaves and soil N mineralization in a controlled environment. In the field, complex N cycling and other factors, including soil fauna, roots, and microclimatic effects, had a stronger influence on available soil N than residue quality.

First record of Trichodina heterodentata (Ciliophora: Trichodinidae) from Arapaima gigas cultivated in Peru

This study characterized morphologically Trichodina heterodentata Duncan, 1977 from cultivated fingerlings of "pirarucu" Arapaima gigas in Peru. Body and gill smears were air-dried at room temperature, impregnated with silver nitrate and/or stained with gomori trichromic. Prevalence was 100%. Trichodina heterodentata was considered a medium-sized trichodinid with mean body diameter of 56.0+ 5.25 (47.3-76.0) μm, denticulate ring 28.21± 2.71 (20-34.7) μm, adhesive disc 45.7±3.8 (37.1-57.3) μm diameter and number of denticles of 20.7± 2.6 (12-24). The present study reports not only the first occurrence of T. heterodentata in Peru but also the first record of this trichodinid infesting A. gigas. Camparative tables of all reports of T. heterodentata are also presented.

Yield of essential oil and safrole content based on fresh and dry biomass of long pepper in the Brazilian Amazon

Long pepper (Piper hispidinervum) is an Amazonian species of commercial interest due to the production of safrole. Drying long pepper biomass to extract safrole is a time consuming and costly process that can also result in the contamination of the material by microorganisms. The objective of this study was to analyze the yield of essential oil and safrole content of fresh and dried biomass of long pepper accessions maintained in the Active Germoplasm Bank of Embrapa Acre, in the state of Acre, Brazil, aiming at selecting genotypes with best performance on fresh biomass to recommend to the breeding program of the species. Yield of essential oil and safrole content were assessed in 15 long pepper accessions. The essential oil extraction was performed by hydrodistillation and analyzed by gas chromatography. A joint analysis of experiments was performed and the means of essential oil yield and safrole content for each biomass were compared by Student's t-test. There was variability in the essential oil yield and safrole content. There was no difference between the types of biomass for oil yield; however to the safrole content there was difference. Populations 9, 10, 12 and 15 had values of oil yield between 4.1 and 5.3%, and safrole content between 87.2 and 94.3%. The drying process does not interfere in oil productivity. These populations have potential for selection to the long pepper breeding program using oil extraction in the fresh biomass

Triterpenes of leaves from Piranhea trifoliata (Picrodendraceae)

ABSTRACT The Amazon forest is rich in plant species diversity, among them,Piranhea trifoliata stands out, which is popularly known as piranheira, because their fruits are eaten by fish. Their barks are used as bath composition on uterus inflammation and as tea in malaria treatment. This study aimed to fractionate the dichloromethane and dichloromethane phase from methanolic extract of leaves of Piranhea trifoliata. The leaves were dried, grounded and extracted with dichloromethane, methanol and water. The methanol extract was partitioned with dichloromethane and ethyl acetate. The chromatographic fractionation yielded six pentacyclic triterpenoids: friedelan-3-one, 28-hydroxy-friedelan-3-one, 30-hydroxy-friedelan-3-one, lupeol, α- and β-amyrin mixture, besides the mixture of the steroids: β-sitosterol and stigmasterol. The substances structures were identified by 1H- and13C-Nuclear Magnetic Resonance (NMR) analysis and literature data comparison. This is the first report describing the chemical study of P. trifoliata leaves.

III - Contribuição para o estudo biológico e ecológico das Podostemaceae do salto do Piracicaba

1 - The Author, in this 3 thd. contribution, concludes the study of the biology and ecology of the species Tristicha trifaria (Willd.) Spreng. and Mourera aspera (Bong.) Tul., both of the Piracicaba Fall. 2 - According to the results of Dr. Peter van Royen (State Herbarium of Leiden, Holland), who made a complete revision of Podostemaceae of the Piracicaba Fall, the species Tristicha hypnoides (St. Hil.) Spreng. var. Hilarii Tul. and Mnioppsis Glazioviana Warm, correspond, respectively, to theTristicha trifaria (Willd.) Spreng. and Mniopsis weddelliana Tul. Apinagia Accorsii Toledo was transferred by Royen to the genus Wettsteiniola. So, its new name is Wettsteiniola accorsii (Toledo) v. Royen. 3 - Propagation by seeds may occur in the following places: a) placenta of partially open fruits; b) external and internal walls of the open capsules; c) pedicels of the fruits; d) remains of rhizomes, branches, etc. e) organic residues accumulated in water holes in the fall; f) clean rocks, in which the little groups of seedlings seems to be a colony of algae. Seeds adhere to the substrata above by means, of a mucilage produced by the transformation of the external integuments in contact with water. 4 - In the growth of the four species below it was found in Piracicaba Fall conspicuous zoning so scattered: a) Wettsteiniola accorsii (Toledo) v. Royen, in rocks situated just within the water fall, where velocity of the current and aeration of the water are very high. b) Tristicha trifaria (Willd.) Spreng. and Mniopsis weddelliana Tul., in rocks at some distance (100 m more or less) upstream until near the bridge across the river. c) Mourera aspera (Bong.) Tul., 300 m upwards the bridge. 5- During 1949, the ecological conditions of the Piracicaba Fall were changed due to the following factors: a) dry season very long, begining from last period of June until 30 november; b) stopping, during four months, of water from the Atibaia river (one of the components of Piracicaba river) near to the city of Americana, in the place where a new station of the Companhia Paulista de Força e Luz was build. In consequence, most of the Podostemaceae died. On the dry rocks there were only fruits and dried plants. 6 - Tristicha trifaria has the same biological and ecological behavior as the Mniopsis weddelliana,. 7 - The vegetative propagation of Tristicha trifaria is made by increasing of its branches, production of stolons with vegetatives buds and regeneration of old parts in especial conditions of water and aeration. 8 - Mourera aspera has the same vegetative propagation as the Wettsteiniola accorsii; it produces stolons (in very little percentage) with vegetative buds, branches of the rhizomes and regeneration of active old parts. 9 - Frequently, there is, on the plants an accumulation of sand, silt, loam, organic substances, and so on. The quantity of material stored depends of the purity of the water, of the morphology of the plants and of the situation on the fall. 10 - In extrem conditions of dry heat, the surviving of the species in its habitat depends exclusively from germination of seeds in the mentioned substrata. Exceptionally, some plants survive in a few water pockets full with the weak remaining current.

Sôbre a determinação do nitrogênio, do fósforo e do potássio no mesmo extrato

The determination of total nitrogen, phosphorus, and potassium in plant material can be carried out in a common extract prepared with sulphuric acid and 30 per cent hydrogen peroxide. Nitrogen is estimated by direct nesslerization of a suitable aliquot (1-5 ml of the 50 ml extract made out of 250 mg of dried material); in order to avoid excessive acidity, 10 ml of Nessler's reagent should be employed. An aliquot of 1-5 ml suffices for the colorimetric determination of phosphorus by the molybdenum method; to reduce the phosphomolybdate complex 2 ml of a 2% SnC12 soln are necessary. Potassium is determined by the cobaltinitrite method after elimination of ammonium salts with the aid of aqua-regia.

Sombreamento dos cafèzais I.: resultados de três ciclos bienais (1953/1958) obtidos na escola "Luiz de Queiroz"

The present paper discusses the data obtained in shade and unshaded coffee plots at Piracicaba, São Paulo, Brazil. The results, analysed statistically, can be summarized as follows: a) unshaded plots produced 17% more than shaded ones; b) the percentage of coffee berry borer infestation was higher in shaded plots as compared with unshaded ones; c) the percentage of green (not ripened fruits) depends of the harvest time. When the harvest was retarded, the percentage of green fruits was higher in the shaded plots. When the percentage of green fruits was the same, both in shaded and unshaded plots, the percentage of ripened was higher and the percentage of dried fruits was lesser in the shaded plots as compared with unshaded ones; d) other comparisons as production of dried grains by the field fruits, relation between dried fruits and dried grains and cup-test by expert coffee-taster, did not show differences among shaded and unshaded plots.

A absorção de nitrogênio, fósforo, potássio, cálcio, magnésio, enxôfre e silício pela cana de açúcar, Co 419, e o seu crescimento em função da idade

This paper describes the data obtained for the growth of sugar cane, Variety Co 419, and the amount and rate of absorption of nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, and silicon, according to the age of the plant, in the soil and climate conditions of the state of S. Paulo, Brazil. An experiment was installed in the Estação Experimental de Cana de Açúcar "Dr. José Vizioli", at Piracicaba, state of S. Paulo, Brazil, and the soil "tèrra-roxa misturada" presented the following composition: Sand (more than 0,2 mm)........................................................................ 8.40 % Fine sand (from 0,2 to less than 0,02 mm)................................................. 24.90 % Silt (from 0,02 to less than 0,002 mm)...................................................... 16.40 % Clay (form 0,002 mm and less)................................................................ 50.20 % pH 10 g of soil and 25 ml of distilled water)..................................................... 5.20 %C (g of carbon per 100 g of soil)................................................................. 1.00 %N (g of nitrogen per 100 g of soil)............................................................... 0.15 P0(4)-³ (me. per 100 g of soil, soluble in 0,05 normal H2SO4) ............................... 0.06 K+ (exchangeable, me. per 100 g of soil)....... 0.18 Ca+² (exchangeable, me. per 100 g of soil)...... 2.00 Mg+² (exchangeable, me. per 100 g of soil)...... 0.66 The monthly rainfall and mean temperature from January 1956 to August 1957 are presented in Table 1, in Portuguese. The experiment consisted of 3 replications of the treatments: without fertilizer and with fertilizer (40 Kg of N, from ammonium sulfate; 100 Kg of P(2)0(5) from superphosphate and 40 Kg K2 O, from potassium chloride). Four complete stools (stalks and leaves) were harvested from each treatment, and the plants separated in stalks and leaves, weighed, dried and analysed every month from 6 up to 15 months of age. The data obtained for fresh and dry matter production are presented in table 2, and in figure land 2, in Portuguese. The curves for fresh and dry matter production showed that fertilized and no fertilized sugar cane with 6 months of age presents only 5% of its total weight at 15 months of age. The most intense period of growth in this experiment is located, between 8 and 12 months of age, that is between December 1956 and April 1957. The dry matter production of sugar cane with 8 and 12 months of age was, respectively, 12,5% and 87,5% of the total weight at 15 months of age. The growth of sugar cane in relation to its age follows a sigmoid curve, according to the figures 1, 2 and 3. The increase of dry matter production promoted by using fertilizer was 62,5% when sugar cane was 15 months of age. The concentration of the elements (tables 4 and 5 in Portuguese) present a general trend of decreasing as the cane grows older. In the stalks this is true for all elements studied in this experiment. But in the leaves, somme elements, like sulfur and silicon, appears to increase with the increasing of age. Others, like calcium and magnesium do not show large variations, and finally a third group, formed by nitrogen, phosphorus and potassium seems to decrease at the beginning and later presents a light increasing. The concentration of the elements was higher in the leaves than in the stalks from 6 up to 15 months of age. There were some exceptions. Potassium, magnesium and sulfur were higher in the stalks than in the leaves from 6 up to 8 or 9 months of age. After 9 months, the leaves presented more potassium, magnesium and sulfur than the stalks. The percentage of nitrogen in the leaves was lower in the plants that received fertilizer than in the plants without fertilizer with 6, 7, 8, 10, 11 and 13 months of age. This can be explained by "dilution effect". The uptake of elements by 4 stools (stalks and leaves) of sugar cane according to the plant age is showed in table 6, in Portuguese. The absorption of all studied elements, nitrogen, phosphorus, potassium, calcium, magnesium, sulfur and silicon, was higher in plants that received fertilizer. The trend of uptake of nitrogen and potassium is similar to the trend of production of dry matter, that is, the maximum absorption of those two nutrients occurs between 9 and 13 months of age. Finaly, the maxima amounts of elements absorbed by 4 stools (stalks and leaves) of sugar cane plants that received fertilizer are condensed in the following table: Element Maximum absorption in grams Age of the plants in months Nitrogen (N) 81.0 14 Phosphorus (P) 6.8 15 Potassium (K) 81.5 15 Calcium (Ca) 19.2 15 Magnesium (Mg) 13.9 13 Sulfur (S) 9.3 15 Silicon (Si) 61.8 15 It is very interesting to note the low absorption of phosphorus even with 100 kg of P2O5 per hectare, aplied as superphosphate. The uptake of phosphorus was lower than calcium, magnesium and sulfur. Also, it is noteworthy the large amount of silicon absorbed by sugar cane.

Substituição de subprodutos de trigo pelo sorgo moído na alimentação de pintos

Searching for a substitute of wheat bran and wheat standard middlings in chick mashes, three experiments were carried out using ground sorghums. In the first one, 30% of Atlas, Kafir e White Afrikan x Sumac (seed chops) were substituted for 30% of wheat by-products. All the rations with sorghum grain gave inferior results. In another experiment, 7, 14, 20 and 30% of sorghum substituted equal percentages of those wheat by-products, the best results having been obtained with 7% of Atlas and 23% of wheat by-products. Finally, in a third experiment, 5% of dried cow manure plus 10, 20 and 30% of ground Atlas sorghum were substituted for 5% of alfalfa hay meal plus, respectively, 10, 20 and 30% of wheat by-products. All results obtained from rations containing sorghum were as good as or better than that given by the ration including alfalfa hay meal and only wheat by-products. Under the conditions of this experiment, 5% of cow manure plus 12,25% of sorghum and 17,75% of wheat by-products is supposed to be the best combination to be recommended, this result having been attained through the study of the regression equation.