Mycobacterium tuberculosis complex differentiation using gyrB-restriction fragment length polymorphism analysis

Mycobacterium tuberculosis complex (MTBC) members are causative agents of human and animal tuberculosis. Differentiation of MTBC members is required for appropriate treatment of individual patients and for epidemiological purposes. Strains from six MTBC species - M. tuberculosis, M. bovis subsp. bovis, M. bovis BCG, M. africanum, M. pinnipedii, and "M. canetti" - were studied using gyrB-restriction fragment length polymorphism (gyrB-RFLP) analysis. A table was elaborated, based on observed restriction patterns and published gyrB sequences. To evaluate applicability of gyrB-RFLP at Instituto Adolfo Lutz, São Paulo, Mycobacterial Reference Laboratory, 311 MTBC clinical isolates, previously identified using traditional methods as M. tuberculosis (306), M. bovis (3), and M. bovis BCG (2), were analyzed by gyrB-RFLP. All isolates were correctly identified by the molecular method, but no distinction between M. bovis and M. bovis BCG was obtained. Differentiation of M. tuberculosis and M. bovis is of utmost importance, because they require different treatment schedules. In conclusion, gyrB-RFLP is accurate and easy-to-perform, with potential to reduce time needed for conventional differentiation methods. However, application for epidemiological studies remains limited, because it cannot differentiate M. tuberculosis from M. africanum subtype II, and "M. canetti", M. africanum subtype I from M. pinnipedii, and. M. bovis from M. bovis BCG.

Mycobacterium avium restriction fragment lenght polymorphism-IS IS1245 and the simple double repetitive element polymerase chain reaction typing method to screen genetic diversity in Brazilian strains

Simple double repetitive element polymerase chain reaction (MaDRE-PCR) and Pvu II-IS1245 restriction fragment length polymorphism (RFLP) typing methods were used to type 41 Mycobacterium avium isolates obtained from 14 Aids inpatients and 10 environment and animals specimens identified among 53 mycobacteria isolated from 237 food, chicken, and pig. All environmental and animals strains showed orphan patterns by both methods. By MaDRE-PCR four patients, with multiple isolates, showed different patterns, suggesting polyclonal infection that was confirmed by RFLP in two of them. This first evaluation of MaDRE-PCR on Brazilian M. avium strains demonstrated that the method seems to be useful as simple and less expensive typing method for screening genetic diversity in M. avium strains on selected epidemiological studies, although with limitation on analysis identical patterns except for one band.

IS6110 restriction fragment length polymorphism typing of Mycobacterium tuberculosis isolates from East Azerbaijan Province of Iran

To investigate the genetic variation among Mycobacterium tuberculosis isolates in the East Azerbaijan Province of Iran and to evaluate the level of and risk factors for recent transmission of tuberculosis (TB), we performed IS6110-based restriction fragment length polymorphism analysis of strains, isolated from 105 patients during the period of September 2002 to March 2003 in TB centers and university hospitals of the province. Among 105 isolates, 81 different IS6110 patterns were found, of which 70 were observed only once and 11 were shared by two to eight isolates. Ninety-six isolates (91.4%) were found to have more than five copies of IS6110 and together with high patterns polymorphism, shows that IS6110-RFLP typing could be useful for studying the epidemiology of TB in Azerbaijan. The minimum estimated rate of recent transmission was 23%, suggesting that the degree of recent transmission in East Azerbaijan Province is relatively low. Clustering was not associated with age, sex or site of infection of TB but drug-resistant isolates were less likely to be clustered than sensitive isolates (p < 0.05).

Novel polymerase chain reaction-restriction fragment length polymorphism assay to determine internal transcribed spacer-2 group in the Chagas disease vector, Triatoma dimidiata (Latreille, 1811)

Triatoma dimidiata is the most important Chagas disease insect vector in Central America as this species is primarily responsible for Trypanosoma cruzi transmission to humans, the protozoan parasite that causes Chagas disease. T. dimidiata sensu lato is a genetically diverse assemblage of taxa and effective vector control requires a clear understanding of the geographic distribution and epidemiological importance of its taxa. The nuclear ribosomal internal transcribed spacer 2 (ITS-2) is frequently used to infer the systematics of triatomines. However, oftentimes amplification and sequencing of ITS-2 fails, likely due to both the large polymerase chain reaction (PCR) product and polymerase slippage near the 5' end. To overcome these challenges we have designed new primers that amplify only the 3'-most 200 base pairs of ITS-2. This region distinguishes the ITS-2 group for 100% of known T. dimidiata haplotypes. Furthermore, we have developed a PCR-restriction fragment length polymorphism (RFLP) approach to determine the ITS-2 group, greatly reducing, but not eliminating, the number of amplified products that need to be sequenced. Although there are limitations with this new PCR-RFLP approach, its use will help with understanding the geographic distribution of T. dimidiata taxa and can facilitate other studies characterising the taxa, e.g. their ecology, evolution and epidemiological importance, thus improving vector control.

Molecular identification of Giardia duodenalis in Ecuador by polymerase chain reaction-restriction fragment length polymorphism

The aim of this study was to determine the genetic diversity of Giardia duodenalis present in a human population living in a northern Ecuadorian rain forest. All Giardia positive samples (based on an ELISA assay) were analysed using a semi-nested polymerase chain reaction-restriction fragment length polymorphism assay that targets the glutamate dehydrogenase (gdh) gene; those amplified were subsequently genotyped using NlaIV and RsaI enzymes. The gdh gene was successfully amplified in 74 of 154 ELISA positive samples; 69 of the 74 samples were subsequently genotyped. Of these 69 samples, 42 (61%) were classified as assemblage B (26 as BIII and 16 as BIV), 22 (32%) as assemblage A (3 as AI and 19 as AII) and five (7%) as mixed AII and BIII types. In this study site we observe similar diversity in genotypes to other regions in Latin America, though in contrast to some previous studies, we found similar levels of diarrheal symptoms in those individuals infected with assemblage B compared with those infected with assemblage A.

Limits of a rapid identification of common Mediterranean sandflies using polymerase chain reaction-restriction fragment length polymorphism

A total of 131 phlebotomine Algerian sandflies have been processed in the present study. They belong to the species Phlebotomus bergeroti, Phlebotomus alexandri, Phlebotomus sergenti, Phlebotomus chabaudi, Phlebotomus riouxi, Phlebotomus perniciosus, Phlebotomus longicuspis, Phlebotomus perfiliewi, Phlebotomus ariasi, Phlebotomus chadlii, Sergentomyia fallax, Sergentomyia minuta, Sergentomyia antennata, Sergentomyia schwetzi, Sergentomyia clydei, Sergentomyia christophersi and Grassomyia dreyfussi. They have been characterised by sequencing of a part of the cytochrome b (cyt b), t RNA serine and NADH1 on the one hand and of the cytochrome C oxidase I of the mitochondrial DNA (mtDNA) on the other hand. Our study highlights two sympatric populations within P. sergenti in the area of its type-locality and new haplotypes of P. perniciosus and P. longicuspis without recording the specimens called lcx previously found in North Africa. We tried to use a polymerase chain reaction-restriction fragment length polymorphism method based on a combined double digestion of each marker. These method is not interesting to identify sandflies all over the Mediterranean Basin.

The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA

Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis: C. para- psilosis sensu stricto, Candida orthopsilosis, andCandida metapsilosis. In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories.

Dietary effects on muscle fatty acid composition of finished heifers

The effects of diet on Longissimus muscle fatty acid composition was determined using 24 crossbred heifers of Simmental vs. Nelore and Limousin vs. Nelore. The experimental diets were: 1) corn and yeast (CY); 2) corn, cottonseed meal + meat and bones meal (CMB); 3) cassava hull and yeast (CHY); 4) cassava hull, cottonseed meal + meat and bones meal (CHMB). Feeding CHMB diets resulted in lower lipid and higher cholesterol contents (P<0.05) for both crosses. Most of the identified fatty acids were monounsaturated, and the highest percentage was found to oleic acid (C18:1w9), with values ranging from 32.54 to 46.42%. Among the saturated fatty acids the palmitic acid (C16:0) showed the highest percentage, with its contents ranging between 19.40 and 32.44%. The highest polyunsaturated/saturated fatty acid ratio was of 0.30, and the lowest was of 0.08. Feeding CY diets resulted in lower cholesterol and higher polyunsaturated fatty acid contents of the Longissimus muscle.

Performance and hormonal profile in broiler chickens fed with different energy levels during post restriction period

The aim of this work was to investigate the influence of diet energy level on performance and hormonal profiles of broilers during post restriction period. It was a split-plot experiment, and the main treatments were in a 2x2 factorial scheme. Birds were fed restricted to 30% of the ad libitum intake, from 7 to 14 days of age. After the restriction period, birds were fed ad libitum with diets containing low (2,900 kcal ME/kg) or high (3,200 kcal ME/kg) energy until 49 days of age. Broilers fed with high energy ration showed lower feed intake and better feed conversion and decreased carcass protein; however, abdominal fat pad, and total carcass fat were not affected by ration energy levels or feeding program. Neither diet energy level nor feed restriction program changed body weight at 49 days. The profile of insulin-like growth factor-1 (IGF-1) was reduced during the feed restriction period, but increased at refeeding period. Feeding program and ration energy level did not affect T3, T4 and growth hormone serum concentrations. Feed restriction at 30% of ad libitum intake is not enough to promote changes on carcass quality, related to fat deposition, and on metabolic hormone levels, except IGF-1 seric level that has rapid increase after feed restriction.

Feeding strategies and energy to protein ratio on tambaqui performance and physiology

The objective of this work was to evaluate the effect of feed deprivation and refeeding with diets containing different energy to protein ratios (E/P) on the performance and physiology of juvenile tambaqui (Colossoma macropomum). A 4x2 factorial arrangement with three replicates was used, with four E/P ratios (11.5, 10.5, 9.5, and 8.5 kcal g-1 digestible energy per protein) and two feeding regimens (with and without deprivation), during 60 days. Fish from the food-deprived group were fasted for 14 days and refed from the fifteenth to the sixtieth day, whereas the remaining fish were fed for 60 days. At the end of the experimental period, weight of fish subjected to food deprivation was lower than that of those continuously fed; however, this condition did not influence the physiological parameters analyzed. Tambaqui fed 11.5 kcal g-1 achieved lower final weight than those fed with the other diets, in both regimens. Among the physiological parameters, only plasma protein presented significant increase in fish fed 8.5 kcal g-1, in both feeding regimens, probably due to the higher dietary protein concentration. These results indicate that fish show a partial compensatory growth, and that 10.5 kcal g-1 can be recommended for the diet of juvenile tambaqui.

DIETARY FIBRE CONTENT, PHENOLIC COMPOUNDS AND ANTIOXIDANT ACTIVITY IN SOURSOPS (Annona muricata L.)

ABSTRACT Healthy eating is associated with the consumption of fruits, which are notable for their beneficial effects on human health. The aim of this study was to evaluate the proximate composition, composition of fibers and components with antioxidant activity in soursops varieties Crioula, Lisa and Morada of physiological maturity (PM) and mature (M). The protein, lipid and moisture contents did not differ between soursop varieties, but the ash contents were higher in the Morada-PM (0.56%±0.03) and the Morada-M (0.82%±0.10) varieties. The Crioula-M variety showed higher levels of total dietary fibre (5.76%±0.12). The Lisa-M variety showed higher levels of insoluble dietary fibre (4.46%±0.00). The Lisa-M variety also showed a higher level of phenolic compounds (284.25 mg gallic acid/100 g of soursop pulp), differing significantly (p <0.05) from the Crioula-PM soursop (154.40 mg of gallic acid/100 g of soursop pulp). Under the DPPH• system, the soursops that showed highest antioxidant activity were the Crioula-M (EC50 of 156.40 g.g DPPH-1) and the Crioula-PM (EC50 of 162.41 g.g DPPH-1), which differed significantly from the Morada soursops. The results suggest that the consumption of soursops is useful for increasing concentrations of bioactive compounds and dietary fibre.

Multidetector-row computed tomography in the preoperative diagnosis of intestinal complications caused by clinically unsuspected ingested dietary foreign bodies: a case series emphasizing the use of volume rendering techniques

Objective The present study was aimed at describing a case series where a preoperative diagnosis of intestinal complications secondary to accidentally ingested dietary foreign bodies was made by multidetector-row computed tomography (MDCT), with emphasis on complementary findings yielded by volume rendering techniques (VRT) and curved multiplanar reconstructions (MPR). Materials and Methods The authors retrospectively assessed five patients with surgically confirmed intestinal complications (perforation and /or obstruction) secondary to unsuspected ingested dietary foreign bodies, consecutively assisted in their institution between 2010 and 2012. Demographic, clinical, laboratory and radiological data were analyzed. VRT and curved MPR were subsequently performed. Results Preoperative diagnosis of intestinal complications was originally performed in all cases. In one case the presence of a foreign body was not initially identified as the causal factor, and the use of complementary techniques facilitated its retrospective identification. In all cases these tools allowed a better depiction of the entire foreign bodies on a single image section, contributing to the assessment of their morphology. Conclusion Although the use of complementary techniques has not had a direct impact on diagnostic performance in most cases of this series, they may provide a better depiction of foreign bodies' morphology on a single image section.

The effect of soy dietary supplement and low dose of hormone therapy on main cardiovascular health biomarkers: a randomized controlled trial

PURPOSE: To assess the effects of a soy dietary supplement on the main biomarkers of cardiovascular health in postmenopausal women compared with the effects of low-dose hormone therapy (HT) and placebo.METHODS: Double-blind, randomized and controlled intention-to-treat trial. Sixty healthy postmenopausal women, aged 40-60 years, 4.1 years mean time since menopause were recruited and randomly assigned to 3 groups: a soy dietary supplement group (isoflavone 90mg), a low-dose HT group (estradiol 1 mg plus noretisterone 0.5 mg) and a placebo group. Lipid profile, glucose level, body mass index, blood pressure and abdominal/hip ratio were evaluated in all the participants at baseline and after 16 weeks. Statistical analyses were performed using the χ2 test, Fisher's exact test, Kruskal-Wallis non-parametric test, analysis of variance (ANOVA), paired Student's t-test and Wilcoxon test.RESULTS: After a 16-week intervention period, total cholesterol decreased 11.3% and LDL-cholesterol decreased 18.6% in the HT group, but both did not change in the soy dietary supplement and placebo groups. Values for triglycerides, HDL-cholesterol, glucose level, body mass index, blood pressure and abdominal/hip ratio did not change over time in any of the three groups.CONCLUSION: The use of dietary soy supplement did not show any significant favorable effect on cardiovascular health biomarkers compared with HT. Clinical Trial Registry: The trial is registered at the Brazilian Clinical Trials Registry (Registro Brasileiro de Ensaios Clínicos - ReBEC), number RBR-76mm75.

Long term dietary deficiency in steers: vital functions and T3 and IGF-1 relationships

To evaluate the influence of diets with different degrees of energy deficiency on the hormonal profile and vital functions, 12 steers were randomly distributed into 3 groups of 4 animals. For 140 days, each group received (G1) a diet to promote a weight gain of 900gr/day (17.7 Mcal/d DE and 13% CP), (G2) 80% of the maintenance requirements (5.8 Mcal/d DE and 7% CP), or (G3) 60% of the maintenance requirements (4.7 Mcal/d DE and 5% CP). In G2 and G3, the energy deficit caused a marked decrease in the heart rate and respiratory rate and a reduction in the blood levels of Insulin like growth factor-1 (IGF-1) and triiodothyronine (T3). The decrease in heart rate, respiratory movement and, to a lesser extent, reduction of the rectal temperature, reflected the low status of energy and was negatively impacted by the low levels of T3. There was a strong correlation between the hormones T3 and IGF-1 (r=0.833). There were also strong correlations between T3 and HR (r=0.701), T3 and RR (r=0.632), IGF-1 and HR (r=0.731), and IGF-1 and RR (r=0.679). There were intermediate correlations between T3 and TºC (r=0.484), T3 and insulin (r=0.506), IGF-1 and insulin (r=0.517), and IGF-1 and TºC (r=0.548). This study showed the influence of a long period of providing an energy-deficient diet on animal performance, correlating hormonal status and vital functions in growing cattle. The results indicated that the evaluated parameters represent an important tool for the early detection of dietary deficiency.

Chemical control of morning glory as a function of water restriction levels

Among the herbicides recommended for the dry season and registered to sugarcane crop, amicarbazone, isoxaflutole and the association diuron + hexazinone + sulfomethuron-methyl can be highlighted. These are pre-emergence herbicides efficient against broad-leaved weeds. Morning glory causes large losses in infested sugarcane fields by bending the stalks and interfering in harvesting. In this study the effectiveness of pre-emergence herbicides for two species of morning glory (Ipomoea hederifolia and Ipomoea grandifolia) was evaluated. Treatments were arranged in completely randomized factorial design (4 x 7). There were four periods of water restriction (0, 30, 60 and 90 days), seven chemical treatments [diuron + hexazinone + sulfometuron-methyl (1387 + 391 + 33.35 g a.i. ha-1), diuron + hexazinone + sulfometuron-methyl (1507.5 + 425 + 36.25 g a.i. ha-1), diuron + hexazinone + sulfometuron-methyl (1658.25 + 467.5 + 39.87 g a.i. ha-1), diuron + hexazinone + sulfometuron­methyl (1809 + 510 + 43.5 g a.i. ha-1), amicarbazone (1190 g a.i. ha-1), amicarbazone + isoxaflutole (840 + 82.5 g a.i. ha-1)] and a control with no application. At 7, 14, 21 and 28 days after the restoration of moisture, control was visually evaluated. After the final evaluation, the dry mass of morning glories was measured. At 90 days of water restriction, diuron + hexazinone + sulfometuron-methyl was more effective to control I. hederifolia than the amicarbazone + isoxaflutole tank mixture. The four diuron + hexazinone + sulfometuron­methyl doses have reduced morning glory dry mass to zero; whereas treatments with amicarbazone have not. The most effective treatment for morning glory control was diuron + hexazinone + sulfometuron-methyl. This result may be due to a possible synergistic interaction.