In vitro effects of extracts and purified tannins of sainfoin (Onobrychis viciifolia) against two cattle nematodes

Sainfoin (Onobrychis viciifolia) is a condensed tannin (CT)-containing legume and has anthelmintic potential against gastrointestinal nematodes of ruminants. This study investigated in vitro effects of acetone/water extracts and derived CT fractions from different types of sainfoin (i.e. accessions) against larvae of Cooperia oncophora and Ostertagia ostertagi by applying the larval feeding inhibition assay (LFIA). Seven sainfoin accessions were extracted and tested with L1 larvae at 10 and 40 μg extract/ml. In addition, CT in extracts from 4 accessions were fractionated according to polymer size and tested by LFIA at two concentrations (2 and 10 μg CT fraction/ml). All sainfoin extracts caused significant inhibition of L1-feeding of both C. oncophora and O. ostertagi with varying intensity compared to the control (phosphate buffered saline). For both nematode species the in vitro effect was positively correlated with CT content in the extracts, but not with any of the structural CT parameters. In contrast, the 16 CT fractions revealed significant correlations between in vitro effect and CT content, polymer size (mean degree of polymerisation, mDP) and monomeric composition (prodelphinidin percentage, % PD). These differences between crude extracts and purified fractions may stem from the fact that extracts contain complex CT mixtures, which may mask and thus suppress CT structural effects. This study provides the first indication that, apart from CT and % PD content, polymer size also contributes to anthelmintic activity of CTs. The results, therefore, suggest that the inter-accession variability in CT content and composition needs to be taken into account in future plant breeding programmes which seek to enhance the anthelmintic properties of sainfoin

Relative populations and toxin production by Aspergillus flavus and Fusarium verticillioides in artificially inoculated corn at various stages of development under field conditions

The presence, development and production of mycotoxins by Aspergillus flavus and Fusarium verticillioides were studied in corn ears under field conditions after artificial contamination of corn silks. The planted area was divided into five treatments: T1, inoculated with A.flavus solution containing 1 x 10(8) spores, ears covered; T2, inoculated with F. verticillioides solution containing 1 X 10(8) spores, ears covered; T3, inoculated with E verticillioides plus A. flavus solution containing 1 x 10(8) spores of each, ears covered; T4, sprayed with sterile phosphate-buffered saline, ears covered; TS, non-sprayed silks, uncovered ears. Soil and air samples were also collected and analysed for the occurrence of fungi. Water activity, relative air humidity, rainfall and temperature were determined to assess the correlation between abiotic factors and the presence of fungi in the samples. Contamination with the inoculated fungus predominated in T1 and T2. In the other treatments, F. verticillioides was the most frequently isolated contaminant irrespective of treatment. Considering the production of mycotoxins, a positive relation between the production of fumonisins B-1 and B-2 and the frequency of F. verticillioides was statistically verified in all treatments. (C) 2007 Society of Chemical Industry.

Use of L-Proline and ATP Production by Trypanosoma cruzi Metacyclic Forms as Requirements for Host Cell Invasion

The process of host cell invasion by Trypanosoma cruzi depends on parasite energy. What source of energy is used for that event is not known. To address this and other questions related to T. cruzi energy requirements and cell invasion, we analyzed metacyclic trypomastigote forms of the phylogenetically distant CL and G strains. For both strains, the nutritional stress experienced by cells starved for 24, 36, or 48 h in phosphate-buffered saline reduced the ATP content and the ability of the parasite to invade HeLa cells proportionally to the starvation time. Inhibition of ATP production by treating parasites with rotenone plus antimycin A also diminished the infectivity. Nutrient depletion did not alter the expression of gp82, the surface molecule that mediates CL strain internalization, but increased the expression of gp90, the negative regulator of cell invasion, in the G strain. When L-proline was given to metacyclic forms starved for 36 h, the ATP levels were restored to those of nonstarved controls for both strains. Glucose had no such effect, although this carbohydrate and L-proline were transported in similar fashions. Recovery of infectivity promoted by L-proline treatment of starved parasites was restricted to the CL strain. The profile of restoration of ATP content and gp82-mediated invasion capacity by L-proline treatment of starved Y-strain parasites was similar to that of the CL strain, whereas the Dm28 and Dm30 strains, whose infectivity is downregulated by gp90, behaved like the G strain. L-Proline was also found to increase the ability of the CL strain to traverse a gastric mucin layer, a property important for the establishment of T. cruzi infection by the oral route. Efficient translocation of parasites through gastric mucin toward the target epithelial cells in the stomach mucosa is an essential requirement for subsequent cell invasion. By relying on these closely associated ATP-driven processes, the metacyclic trypomastigotes effectively accomplish their internalization.

Leishmania amazonensis: Biosynthesis of polyprenols of 9 isoprene units by amastigotes

The isoprenoid metabolic pathway in protozoa of the Leishmania genus exhibits distinctive characteristics. These parasites, as well as other members of the Trypanosomatidae family, synthesize ergosterol, instead of cholesterol, as the main membrane sterol lipid. Leishmania has been shown to utilize leucine, instead of acetate as the main precursor for sterol biosynthesis. While mammalian dolichols are molecules containing 15-23 isoprene units, Leishmania amazonensis promastigotes synthesize dolichol of 11 and 12 units. In this paper, we show that the intracellular stages of L. amazonensis, amastigotes, synthesize mainly polyprenols of 9 isoprene units, instead of dolichol. (c) 2007 Elsevier Inc. All rights reserved.

Occurrence of Cryptosporidium (Apicomplexa, Cryptosporidiidae) in Crotalus durissus terrificus (Serpentes, Viperidae) in Brazil

The objective of the present study was to investigate the prevalence of Cryptosporidium (Apicomplexa, Cryptosporidiidae) in the snake Crotalus durissus terrificus (Serpentes, Viperidae). Fifty animals were evaluated for the presence of oocysts of Cryptosporidium sp. at the time of arrival and 30 and 60 days later. Intestinal washings with saline solution (1% body weight), fecal samples, and organ scrapings were collected during the study. Oocysts were concentrated by an ether-phosphate-buffered saline sedimentation technique and then separated by a density gradient centrifugation technique. Smears were made with the sediment and submitted to modified acid-fast and auramine-rhodamine staining. Cryptosporidium-positive smears were used as controls for the experimental findings. The overall prevalence of Cryptosporidium sp. oocysts was 14%. Among the positive snakes, oocysts were detected only in the intestinal washing in two specimens, only in the feces in four specimens, and in both materials at least once in one specimen. The positive snakes were predominantly from Santa Maria da Serra city State of São Paulo (57.1%). We also observed that all of the examinations that presented positive results were obtained at least 27 days after the capture of the animals.

Serum protein concentrations in calves with experimentally induced pneumonic pasteurellosis

Foram examinados 10 bezerros da raça Holandesa com duas a quatro semanas de idade, distribuídos aleatoriamente em dois grupos, controle e infectado. Os bezerros do grupo-controle foram inoculados por via intrabronquial com 5ml de solução salina fosfato-tamponada Dulbecco (DPBSS). Os do grupo infectado receberam um inóculo contendo 5x10(9) unidades formadoras de colônia-UFC de Mannheimia (Pasteurella) haemolytica, suspensas em 5ml de DPBSS. As amostras de sangue foram colhidas 15 minutos antes e uma, duas, quatro e seis horas após a inoculação. O proteinograma sérico foi obtido por eletroforese em gel de acrilamida. As concentrações séricas das proteínas de pesos moleculares 125.000 D (ceruloplasmina), 60.000 D (a1-antitripsina), 45.000 D (haptoglobina) e 40.000 D (glicoproteína ácida) foram significativamente maiores em bezerros infectados do que nos do grupo-controle. Os resultados indicam que as concentrações das proteínas de fase aguda se elevaram mais rapidamente que o previamente imaginado. O proteinograma sérico pode ser útil no monitoramento da evolução da pneumonia de bezerros causada por M. haemolytica.

Estudo clínico e laboratorial da pneumonia de bezerros induzida pela inoculação intrabronquial de Mannheimia haemolytica

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Cytotoxicity of chlorhexidine digluconate to murine macrophages and its effect on hydrogen peroxide and nitric oxide induction

Chlorhexidine, even at low concentrations, is toxic for a variety of eukaryotic cells; however, its effects on host immune cells are not well known. We evaluated in vitro chlorhexidine-induced cytotoxicity and its effects on reactive oxygen/nitrogen intermediate induction by murine peritoneal macrophages. Thioglycollate-induced cells were obtained from Swiss mice by peritoneal lavage with 5 ml of 10 mM phosphate-buffered saline, washed twice and resuspended (10(6) cells/ml) in appropriate medium for each test. Cell preparations contained more than 95% macrophages. The cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay and the presence of hydrogen peroxide (H2O2) and nitric oxide (NO) by the horseradish peroxidase-dependent oxidation of phenol red and Griess reaction, respectively. The midpoint cytotoxicity values for 1- and 24-h exposures were 61.12 ± 2.46 and 21.22 ± 2.44 µg/ml, respectively. Chlorhexidine did not induce synthesis or liberation of reactive oxygen/nitrogen intermediates. When macrophages were treated with various sub-toxic doses for 1 h (1, 5, 10, and 20 µg/ml) and 24 h (0.5, 1, and 5 µg/ml) and stimulated with 200 nM phorbol myristate acetate (PMA) solution, the H2O2 production was not altered; however, the NO production induced by 10 µg/ml lipopolysaccharide (LPS) solution varied from 14.47 ± 1.46 to 22.35 ± 1.94 µmol/l and 13.50 ± 1.42 to 20.44 ± 1.40 µmol/l (N = 5). The results showed that chlorhexidine has no immunostimulating activity and sub-toxic concentrations did not affect the response of macrophages to the soluble stimulus PMA but can interfere with the receptor-dependent stimulus LPS.

An in vitro growth inhibition test for measuring the potency of Leptospira spp. Sejroe group vaccine in buffaloes

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

Improvement in embryo recovery using double uterine flushing

The objective of the present study was to evaluate the effects of double uterine flushing on the recovery of embryos/ova in cattle. Two hundred and ten embryo recovery procedures were conducted using a double uterine flushing method, and the results were compared with 432 conventional single-flushing procedures. Cyclic Limousin (n = 403) and Guzera (n = 239) donor cows received an intravaginal progesterone releasing device and 2 mg of estradiol benzoate on Day 0. Between Days 5 and 9, donors received decreasing doses of FSH, which ranged from 200 to 300 IU (Bos indicus) and 300 to 500 IU (Bos taut-us). on the afternoon of Day 7, donors received an injection of 500 mu g cloprostenol and progesterone implants were removed 12 It later (morning of Day 8). Artificial insemination was performed between 14 and 26 h after first detection of behavioral estrus. Cows were randomly assigned to have embryos recovered by a double-flushing method (n = 210) or the conventional single-flushing procedure (n = 432). For the double-flushing procedure, after first flushing the whole uterus with 1 L of Dubelco's Phosphate Buffered Saline (DPBS), a Foley catheter was positioned in the uterine body to permit refilling of the uterus with fresh DPBS (80-150 mL). The catheter was closed with the plunger of a disposable 5 mL syringe, and the donors were allowed to rest in a holding area for 30 min. Thereafter, a second flush was performed to recover the solution remaining in the uterus. Animals from the control group were subjected to a single uterine flush. From 2 10 double-flushing procedures, 1409 viable embryos were recovered. In comparison, from 432 cows receiving the single-flushing procedure, 1993 embryos were recovered. Double flushing increased (P < 0.05) the number of embryos recovered per procedure compared to single flushing (6.7 +/- 0.4 versus 4.6 +/- 0.2, respectively; mean +/- S.E.M.). When double flushing was performed, average recovered embryos/ova increased (P < 0.05) from 8.3 +/- 0.4 to 12.7 +/- 0.7 in Limousin and from 7.9 to 11.5 in Guzera. Also, utilization of double flushing increased (P < 0.05) the number of viable embryos from 4.7 +/- 0.3 to 6.9 +/- 0.5 in Limousin and from 4.5 +/- 0.4 to 6.4 +/- 0.7 in Guzera. Mean total embryos/ova was similar (P > 0.05) between the control group and after the first uterine flushing in the double-flushing group; therefore, both flushings were conducted efficiently. In conclusion, double uterine flushing increased embryo recovery in cattle. (C) 2004 Elsevier B.V. All rights reserved.

In vitro cytotoxicity of five glass-ionomer cements

To evaluate the cytotoxic effects of five glass-ionomer cements (GICs) on an odontoblast cell line (MDPC-23), disks of every material were prepared and divided into Group 1: Vitrebond, Group 2: Vitremer, Group 3: Fuji IILC, Group 4: Fuji IX GP, Group 5: Ketac-Molar, Group 6: Z-100 (positive control). In Group 7, phosphate-buffered saline solution (negative control) was applied on filter paper. After placing the samples in the bottom of wells, the cells (30,000 cells/cm(2)) were plated and incubated for 72 h. The cell number was counted, the cell morphology was assessed by scanning electron microscopy and the cell metabolism was evaluated using methyltetrazolium assay. The statistical analysis of Kruskal-Wallis was used to determine if the scores obtained for the cell metabolism and number of cells were different at the 95% confidence level. In groups 1, 2, 3, 4, 5, and 6 the materials decreased the cell number by 74.5% 75.5%, 45.5%, 29.5%, 32.5%, and 88.5%, respectively. In groups 1, 2, 3, 4, and 5, the experimental GICs reduced the cell metabolism by 79%, 84%, 54%, 40%, and 42.5%, respectively. Despite the fact that all experimental materials were cytotoxic to the MDPC-23 cells, the GICs were the least cytotoxic. on the other hand, the RMGICs caused the highest cytophatic effects. (C) 2003 Elsevier B.V. Ltd. All rights reserved.

Antimicrobial activity of Calendula officinalis, Camellia sinensis and chlorhexidine against the adherence of microorganisms to sutures after extraction of unerupted third molars

Objective: The objective of this study was to compare the antimicrobial effect of mouthwashes containing Calendula officinalis L., Camellia sinensis (L.) Kuntze and 0.12% chlorhexidine digluconate on the adherence of microorganisms to suture materials after extraction of unerupted third molars. Material and Methods: Eighteen patients with unerupted maxillary third molars indicated for extraction were selected (n=6 per mouthwash). First, the patients were subjected to extraction of the left tooth and instructed not to use any type of antiseptic solution at the site of surgery (control group). After 15 days, the right tooth was extracted and the patients were instructed to use the Calendula officinalis, Camellia sinensis or chlorhexidine mouthwash during 1 week (experimental group). For each surgery, the sutures were removed on postoperative day 7 and placed in sterile phosphate-buffered saline. Next, serial dilutions were prepared and seeded onto different culture media for the growth of the following microorganisms: blood agar for total microorganism growth; Mitis Salivarius bacitracin sucrose agar for mutans group streptococci; mannitol agar for Staphylococcus spp.; MacConkey agar for enterobacteria and Pseudomonas spp., and Sabouraud dextrose agar containing chloramphenicol for Candida spp. The plates were incubated during 24-48 h at 37 degrees C for microorganism count (CFU/nnL). Results: The three mouthwashes tested reduced the number of microorganisms adhered to the sutures compared to the control group. However, significant differences between the control and experimental groups were only observed for the mouthwash containing 0.12% chlorhexidine digluconate. Conclusions: Calendula officinalis L. and Camellia sinensis (L.) Kuntze presented antimicrobial activity against the adherence of microorganisms to sutures but were not as efficient as chlorhexidine digluconate.

Indole ring oxidation by activated leukocytes prevents the production of hypochlorous acid

Hypochlorous acid (HOCl) released by activated leukocytes has been implicated in the tissue damage that characterizes chronic inflammatory diseases. In this investigation, 14 indole derivatives, including metabolites such as melatonin, tryptophan and indole-3-acetic acid, were screened for their ability to inhibit the generation of this endogenous oxidant by stimulated leukocytes. The release of HOCl was measured by the production of taurine-chloramine when the leukocytes (2 x 10(6) cells/mL) were incubated at 37ºC in 10 mM phosphate-buffered saline, pH 7.4, for 30 min with 5 mM taurine and stimulated with 100 nM phorbol-12-myristate acetate. Irrespective of the group substituted in the indole ring, all the compounds tested including indole, 2-methylindole, 3-methylindole, 2,3-dimethylindole, 2,5-dimethylindole, 2-phenylindole, 5-methoxyindole, 6-methoxyindole, 5-methoxy-2-methylindole, melatonin, tryptophan, indole-3-acetic acid, 5-methoxy-2-methyl-3-indole-acetic acid, and indomethacin (10 µM) inhibited the chlorinating activity of myeloperoxidase (MPO) in the 23-72% range. The compounds 3-methylindole and indole-3-acetic acid were chosen as representative of indole derivatives in a dose-response study using purified MPO. The IC50 obtained were 0.10 ± 0.03 and 5.0 ± 1.0 µM (N = 13), respectively. These compounds did not affect the peroxidation activity of MPO or the production of superoxide anion by stimulated leukocytes. By following the spectral change of MPO during the enzyme turnover, the inhibition of HOCl production can be explained on the basis of the accumulation of the redox form compound-II (MPO-II), which is an inactive chlorinating species. These results show that indole derivatives are effective and selective inhibitors of MPO-chlorinating activity.

Lipid peroxidation, antioxidant enzymes and glutathione levels in human erythrocytes exposed to colloidal iron hydroxide in vitro

The free form of the iron ion is one of the strongest oxidizing agents in the cellular environment. The effect of iron at different concentrations (0, 1, 5, 10, 50, and 100 µM Fe3+) on the normal human red blood cell (RBC) antioxidant system was evaluated in vitro by measuring total (GSH) and oxidized (GSSG) glutathione levels, and superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px) and reductase (GSH-Rd) activities. Membrane lipid peroxidation was assessed by measuring thiobarbituric acid reactive substance (TBARS). The RBC were incubated with colloidal iron hydroxide and phosphate-buffered saline, pH 7.45, at 37oC, for 60 min. For each assay, the results for the control group were: a) GSH = 3.52 ± 0.27 µM/g Hb; b) GSSG = 0.17 ± 0.03 µM/g Hb; c) GSH-Px = 19.60 ± 1.96 IU/g Hb; d) GSH-Rd = 3.13 ± 0.17 IU/g Hb; e) catalase = 394.9 ± 22.8 IU/g Hb; f) SOD = 5981 ± 375 IU/g Hb. The addition of 1 to 100 µM Fe3+ had no effect on the parameters analyzed. No change in TBARS levels was detected at any of the iron concentrations studied. Oxidative stress, measured by GSH kinetics over time, occurs when the RBC are incubated with colloidal iron hydroxide at concentrations higher than 10 µM of Fe3+. Overall, these results show that the intact human RBC is prone to oxidative stress when exposed to Fe3+ and that the RBC has a potent antioxidant system that can minimize the potential damage caused by acute exposure to a colloidal iron hydroxide in vitro.

An experimental inoculation system to study citrus-Xylella fastidiosa interactions

Difficulties in reproducing the citrus variegated chlorosis (CVC) disease symptoms in expertmental plants have delayed implementation of studies to better understand the essential aspects of this important disease. In an extensive Study, cultivars of sweet orange (Citrus sinensis) were inoculated with Xylella fastidiosa using procedures that included root immersion, and stein absorption, pricking, or infiltration of the inoculum into plants of different ages. Inoculum consisted of 5-day-old cultures or cell suspensions of CVC strain 9a5c diluted in phosphate-buffered saline. Inoculated plants and controls were grown, or transferred just after inoculation, to 5-liter pots or 72-cell foam trays. Approximately 4, 5, 9, and 12 months after inoculation, leaves were collected and processed for polymerase chain reaction analysis or X. fastidiosa isolation on BCYE agar medium. Root immersion and stem inoculation of 4- and 6-month-old plants resulted in low percentages of symptomatic (0 to 7%) and plants positive by isolation (0 to 9%). Pinpricked or injected stems of I-month-old seedlings resulted in high percentages of plants symptomatic (29 and 90% in Pera Rio, 75, 59, and 83% in Valencia, and 77% in Natal) or positive by isolation (26 and 93% in Pera Rio, 98, 96, and 83% in Valencia, and 77% in Natal), In foam trays, the seedlings grew less, the incubation period was shorter. and disease severity was higher than in pots. This system allows testing of higher numbers of plants in a reduced space with a more precise reproduction of the experimental conditions.