Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6)

HHV-6 is the etiological agent of Exanthem subitum which is considered the sixth most frequent disease in infancy. In immuno-compromised hosts, reactivation of latent HHV-6 infection may cause severe acute disease. We developed a Sybr Green Real Time PCR for HHV-6 and compared the results with nested conventional PCR. A 214 pb PCR derived fragment was cloned using pGEM-T easy from Promega system. Subsequently, serial dilutions were made in a pool of negative leucocytes from 10-6 ng/µL (equivalent to 2465.8 molecules/µL) to 10-9 (equivalent to 2.46 molecules/µL). Dilutions of the plasmid were amplified by Sybr Green Real Time PCR, using primers HHV3 (5' TTG TGC GGG TCC GTT CCC ATC ATA 3)'and HHV4 (5' TCG GGA TAG AAA AAC CTA ATC CCT 3') and by conventional nested PCR using primers HHV1 (outer): 5'CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (outer): 5' ACA TCT ATA ATT TTA GAC GAT CCC 3'; HHV3 (inner) and HHV4 (inner) 3'. The detection threshold was determined by plasmid serial dilutions. Threshold for Sybr Green real time PCR was 24.6 molecules/µL and for the nested PCR was 2.46 molecules/µL. We chose the Real Time PCR for diagnosing and quantifying HHV-6 DNA from samples using the new Sybr Green chemistry due to its sensitivity and lower risk of contamination.

Antiviral activity of the green marine alga Ulva fasciata on the replication of human metapneumovirus

We evaluated the antiviral activity of the marine alga, Ulva fasciata, collected from Rasa beach and Forno beach, Búzios, Rio de Janeiro, Brazil on the replication of human metapneumovirus (HMPV). The algae extracts were prepared using three different methodologies to compare the activity of different groups of chemical composites obtained through these different methodologies. Four out of the six extracts inhibited nearly 100% of viral replication. The results demonstrated that the majority of the extracts (five out of six) possess virucidal activity and therefore have the ability to interact with the extracellular viral particles and prevent the infection. On the other hand, only two extracts (from Forno beach, obtained by maceration and maceration of the decoction) were able to interact with cell receptors, hindering the viral entry. Finally, only the extract of algae collected at Forno beach, obtained by maceration presented intracellular activity. To our knowledge, this is a pioneer study on antiviral activity of marine algae against HMPV. It is also the first on antiviral activity against HMPV ever done in Brazil. The study also shows the effect of different environment factors and different chemical procedures used to obtain the extract on its biological properties.

MULTIPLEX SYBR® GREEN-REAL TIME PCR (qPCR) ASSAY FOR THE DETECTION AND DIFFERENTIATION OF Bartonella henselae AND Bartonella clarridgeiae IN CATS

A novel SYBR® green-real time polymerase chain reaction (qPCR) was developed to detect two Bartonellaspecies, B. henselae and B. clarridgeiae, directly from blood samples. The test was used in blood samples obtained from cats living in animal shelters in Southern Brazil. Results were compared with those obtained by conventional PCR targeting Bartonella spp. Among the 47 samples analyzed, eight were positive using the conventional PCR and 12 were positive using qPCR. Importantly, the new qPCR detected the presence of both B. henselae and B. clarridgeiae in two samples. The results show that the qPCR described here may be a reliable tool for the screening and differentiation of two important Bartonella species.

ANTIFUNGAL ACTIVITY OF SILVER NANOPARTICLES OBTAINED BY GREEN SYNTHESIS

Silver nanoparticles (AgNPs) are metal structures at the nanoscale. AgNPs have exhibited antimicrobial activities against fungi and bacteria; however synthesis of AgNPs can generate toxic waste during the reaction process. Accordingly, new routes using non-toxic compounds have been researched. The proposal of the present study was to synthesize AgNPs using ribose as a reducing agent and sodium dodecyl sulfate (SDS) as a stabilizer. The antifungal activity of these particles against C. albicans and C. tropicalis was also evaluated. Stable nanoparticles 12.5 ± 4.9 nm (mean ± SD) in size were obtained, which showed high activity against Candida spp. and could represent an alternative for fungal infection treatment.

Larvicidal activity of the methanol extract and fractions of the green fruits of Solanum lycocarpum(Solanaceae) against the vector Culex quinquefasciatus (Diptera: Culicidae)

IntroductionThe larvicidal activity of Solanum lycocarpumagainst Culex quinquefasciatus is unknown.MethodsWe evaluated the larvicidal activity of extracts of the green fruits of Solanum lycocarpum against third and fourth instar larvae of C. quinquefasciatus.ResultsDichloromethane and ethyl acetate fractions showed the greatest larvicidal effect at 200mg/L (83.3% and 86.7%, respectively). The methanol and dichloromethane, ethyl acetate, and hydromethanolic fractions demonstrated larvicidal effects against C. quinquefasciatus, with LC50 values of 126.24, 75.13, 83.15, and 207.05mg/L, respectively.ConclusionsThus, when considering new drugs with larvicidal activity from natural products, S. lycocarpum fruits may be good candidate sources.

Transport of Pirarucu Arapaima gigas juveniles in plastic bag

This study examined the stress response of pirarucu juveniles in a closed system transport. Pirarucu (Arapaima gigas) is a native Amazonian fish species from the Osteoglossidae family and an obligated air breather. A short duration transport trial (6h) was undertaken comparing closed polyethylene bags filled with atmospheric air (Air group) and bags filled with pure oxygen (Oxi group). Dissolved oxygen was the only water parameter that presented a difference between fish groups, and was saturated in the oxi group as expected. There was no mortality in either group after transport. Fish feeding was observed 36 h after transport for all fish, and normal feeding consumption was observed at 72 h. In both groups physiological responses were similar. Cortisol did not show any significant alteration during the sampled period. Unlike most fish species, cortisol values were unaltered in both groups during sampling, while glucose presented a significant change up to 12 h after transport. The results showed that pirarucu transport in plastic bags could be made with either atmospheric air or pure oxygen, since physiological response to stress, water quality and feeding behavior after 36 h were similar in both groups.

Effects of clove oil on the stress response of matrinxã (Brycon cephalus) subjected to transport

Fish transport is one of the most stressful procedures in aquaculture facilities. The present work evaluated the stress response of matrinxã to transportation procedures, and the use of clove oil as an alternative to reduce the stress response to transport in matrinxã (Brycon cephalus). Clove oil solutions were tested in concentrations of 0, 1, 5 and 10 mg/L during matrinxã transportation in plastic bags, supplied with water and oxygen as the usual field procedures in Brazil. Clove oil reduced some of the physiological stress responses (plasma cortisol, glucose and ions) that we measured. The high energetic cost to matrinxã cope with the transport stress was clear by the decrease of liver glycogen after transport. Our results suggest that clove oil (5 mg/l) can mitigate the stress response in matrinxã subjected to transport.

Sodium chloride added to transport water and physiological responses of Matrinxã Brycon amazonicus (Teleost: Characidae)

The addition of salt to the water has been used to mitigate stress and improve survival in fishes. This study investigated the effects of sodium chloride (0.0, 1.0, 3.0 and 6.0 g/l) on levels of plasma cortisol, glucose, tryacilglycerol, total protein, hematocrit, hemoglobin, erythrocyte number, liver glycogen and lipid, and muscle lipid in adult matrinxã(Brycon amazonicum) after a 4-h transport and during a 96-h recovery period. Fish were sampled before and after transport, and 24 and 96 h of the recovery period. Plasma cortisol was higher than initial condition immediately after transportation, except in fish transported in 3.0 and 6.0 g/l of salt. A similar pattern was observed for blood glucose but fish transported in water with 0.0, 1.0 and 3.0 g/l of salt needed more than 24 h to return to the initial condition. Liver glycogen was lower after transport in fish not exposed to salt. Hemoglobin, erythrocyte number, total plasma protein and liver lipid did not change during the experiment but hematocrit was lower after transport in all treatments and returned to pre-transport values in 24 h. Reductions of muscle lipid and plasma tryacilglycerol were observed during the recovery period in fish from all treatments. The results show that 6.0 g/l NaCl added to the transport water reduce the stress responses and a 96-h recovery period is needed if no salt is used to mitigate the stress.

Cytochemical characterization of gill and hepatopancreatic cells of the crab Ucides cordatus (Crustacea, Brachyura) validated by cell metal transport

Ucides cordatus (Linnaeus, 1763) is a hypo-hyper-regulating mangrove crab possessing gills for respiratory and osmoregulatory processes, separated in anterior and posterior sections. They also have hepatopancreas, which is responsible for digestion and absorption of nutrients and detoxification of toxic metals. Each of these organs has specific cells that are important for in vitro studies in cell biology, ion and toxic metals transport. In order to study and characterize cells from gills and hepatopancreas, both were separated using a Sucrose Gradient (SG) from 10 to 40% and cells in each gradient were characterized using the vital mitochondrial dye DASPEI (2-(4-dimethylaminostyryl)-N- ethylpyridinium iodide) and Trichrome Mallory's stain. Both in 20 and 40% SG for gill cells and 30% SG for hepatopancreatic cells, a greater number of cells were colored with DASPEI, indicating a larger number of mitochondria in these cells. It is concluded that the gill cells present in 20% and 40% SG are Thin cells, responsible for respiratory processes and Ionocytes responsible for ion transport, respectively. For hepatopancreatic cells, the 30% SG is composed of Fibrillar cells that possess larger number of membrane ion and nutrient transporters. Moreover, the transport of toxic metal cadmium (Cd) by isolated hepatopancreatic cells was performed as a way of following cell physiological integrity after cell separation and to study differences in transport among the cells. All hepatopancreatic cells were able to transport Cd. These findings are the first step for further work on isolated cells of these important exchange epithelia of crabs, using a simple separation method and to further develop successful in vitro cell culture in crabs.

Sarcopromusca pruna (Diptera: Muscidae) as an egg transport host of Dermatobia hominis (Diptera: Cuterebridae) in the cacau region of Bahia, Brazil

Sarcopromusca pruna appears to be the predominant transport host for Dermatobia hominis eggs among cattle herds in central eastern Bahia, Brazil. In the study area, two seasonal peaks of S. Pruna abundance coincide with those of Dermatobia, from mid July through late September and from mid November until early January, two periods of moderate monthly rainfall between anual extremes. Among more than 26,000 flies examined during the study, 75 (all female S. pruna) bore Dermatobia eggs. Certain aspects of Dermatobia behavior and ovoposition habits in the field are also discussed.

Transport pathways in the malaria-infected erythrocyte: characterization and their use as potential targets for chemotherapy

The intraerythrocytic malarial parasite is involved in an extremely intensive anabolic activity while it resides in its metabolically quiescent host cell. The necessary fast uptake of nutrients and the discharge of waste product, are guaranteed by parasite-induced alterations of the constitutive transporters of the host cell and the production of new parallel pathways. The membrane of the host cell thus becomes permeable to phospholipids, purine bases and nucleosides, small non-electrolytes, anions and cations. When the new pathways are quantitatively unimportant, classical inhibitors of native transporters can be used to inhibit parasite growth. Several compounds were found to effectively inhibit the new pathways and consequently, parasite growth. The pathways have also been used to introduce cytotoxic agents. The parasitophorous membrane consists of channels which are highly permeable to small solutes and display no ion selectivity. Transport of some cations and anions across the parasite membrane is rapid and insensitive to classical inhibitors, and in some cases it is mediated by specific antiporters which respond to their respective inhibitors. Macromolecules have been shown to reach the parasitophorous space through a duct contiguous with the host cell membrane, and subsequently to be endocytosed at the parasite membrane. The simultaneous presence of the parasitophorous membrane channels and the duct, however, is incompatible with experimental evidences. No specific inhibitors were found as yet that would efficiently inhibit transport through the channels or the duct.

Green propolis phenolic compounds act as vaccine adjuvants, improving humoral and cellular responses in mice inoculated with inactivated vaccines

Adjuvants play an important role in vaccine formulations by increasing their immunogenicity. In this study, the phenolic compound-rich J fraction (JFR) of a Brazilian green propolis methanolic extract stimulated cellular and humoral immune responses when co-administered with an inactivated vaccine against swine herpesvirus type 1 (SuHV-1). When compared to control vaccines that used aluminium hydroxide as an adjuvant, the use of 10 mg/dose of JFR significantly increased (p < 0.05) neutralizing antibody titres against SuHV-1, as well as the percentage of protected animals following SuHV-1 challenge (p < 0.01). Furthermore, addition of phenolic compounds potentiated the performance of the control vaccine, leading to increased cellular and humoral immune responses and enhanced protection of animals after SuHV-1 challenge (p < 0.05). Prenylated compounds such as Artepillin C that are found in large quantities in JFR are likely to be the substances that are responsible for the adjuvant activity.

Rapid detection of multidrug-resistant Mycobacterium tuberculosis using the malachite green decolourisation assay

Early detection of drug resistance in Mycobacterium tuberculosis isolates allows for earlier and more effective treatment of patients. The aim of this study was to investigate the performance of the malachite green decolourisation assay (MGDA) in detecting isoniazid (INH) and rifampicin (RIF) resistance in M. tuberculosis clinical isolates. Fifty M. tuberculosis isolates, including 19 multidrug-resistant, eight INH-resistant and 23 INH and RIF-susceptible samples, were tested. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and agreement of the assay for INH were 92.5%, 91.3%, 92.5%, 91.3% and 92%, respectively. Similarly, the sensitivity, specificity, PPV, NPV and agreement of the assay for RIF were 94.7%, 100%, 100%, 96.8% and 98%, respectively. There was a major discrepancy in the tests of two isolates, as they were sensitive to INH by the MGDA test, but resistant by the reference method. There was a minor discrepancy in the tests of two additional isolates, as they were sensitive to INH by the reference method, but resistant by the MGDA test. The drug susceptibility test results were obtained within eight-nine days. In conclusion, the MGDA test is a reliable and accurate method for the rapid detection of INH and RIF resistance compared with the reference method and the MGDA test additionally requires less time to obtain results.

Study of the antimalarial properties of hydroxyethylamine derivatives using green fluorescent protein transformed Plasmodium berghei

A rapid decrease in parasitaemia remains the major goal for new antimalarial drugs and thus, in vivo models must provide precise results concerning parasitaemia modulation. Hydroxyethylamine comprise an important group of alkanolamine compounds that exhibit pharmacological properties as proteases inhibitors that has already been proposed as a new class of antimalarial drugs. Herein, it was tested the antimalarial property of new nine different hydroxyethylamine derivatives using the green fluorescent protein (GFP)-expressing Plasmodium bergheistrain. By comparing flow cytometry and microscopic analysis to evaluate parasitaemia recrudescence, it was observed that flow cytometry was a more sensitive methodology. The nine hydroxyethylamine derivatives were obtained by inserting one of the following radical in the para position: H, 4Cl, 4-Br, 4-F, 4-CH3, 4-OCH3, 4-NO2, 4-NH2 and 3-Br. The antimalarial test showed that the compound that received the methyl group (4-CH3) inhibited 70% of parasite growth. Our results suggest that GFP-transfected P. berghei is a useful tool to study the recrudescence of novel antimalarial drugs through parasitaemia examination by flow cytometry. Furthermore, it was demonstrated that the insertion of a methyl group at the para position of the sulfonamide ring appears to be critical for the antimalarial activity of this class of compounds.