The daily variations of airborne fungal spores in Mexico City

The daily and seasonal distribution of airborne fungal particles was recorded in a high altitude tropical zone. Sampling was carried out in the southern part of Mexico City. An Andersen air sampler was used over a period of six months. Ten minutes sampling for each set of plates was done at fixed schedule: 07:30, 14:00 and 19:00 hours. The sampler was placed 10 m above the ground. Daily variation was found to be associated with the season, weather and atmospheric stability. The highest value of mold counts (3195 CFU m⁻³) was recorded in the evening on October, a transitional month between the rainy and the dry seasons, the lowest (45 CFU m⁻³) at noon during the rainy season. Mold counts were significantly correlated with temperature, having negative signs both in the morning and at noon, and being positive in the evening. The abundance of only three genera was recorded.Cladosporium, was isolated more frequently, and its abundance at 14:00 h was of 38%;Alternaria represented 4.0%, at 14:00 h, andAspergillus 3.0% at 7:30 h. Fifteen species belonging to the latter genera were identified and most of them are considered as opportunistic molds of clinical significance.     

Quality Assessment of Bee Pollen-Honey Mixtures Using Thin-Layer Chromatography in Combination with Chemometrics

The aim of this study is to develop a rapid, effect-directed screening method for quality assessment of bee pollen-honey mixtures. The comparative antioxidant potential and phenolic content of honey, bee pollen, and the bee pollen-honey mixtures, was performed using spectrophotometry. The total phenolic content and antioxidative activity of bee pollen-honey mixtures with 20 % bee pollen share were in the range 3.03–3.11 mg GAE/g, and 6.02–6.96 mmol TE/kg, respectively, while mixtures with 30 % bee pollen share contained 3.92–4.18 mg GAE/g, and 9.69–10.11 mmol TE/kg. Chromatographic fingerprint of bee pollen-honey mixtures was performed by high-performance thin-layer chromatography with conditions developed by authors and reported for the first time. Fingerprint analysis hyphenated with chemometrics enabled authenticity assessments of honey in mixtures. Results indicate that bee pollen-honey mixtures represent a food with highly, both, nutritious characteristics and health-promoting effect.     

Synthesis of 4-methylumbelliferyl α-d-mannopyranosyl-(1→6)-β-d-mannopyranoside and development of a coupled fluorescent assay for GH125 exo-α-1,6-mannosidases

Certain bacterial pathogens possess a repertoire of carbohydrate processing enzymes that process host N-linked glycans and many of these enzymes are required for full virulence of harmful human pathogens such as Clostridium perfringens and Streptococcus pneumoniae. One bacterial carbohydrate processing enzyme that has been studied is the pneumococcal virulence factor SpGH125 from S. pneumoniae and its homologue, CpGH125, from C. perfringens. These exo-α-1,6-mannosidases from glycoside hydrolase family 125 show poor activity toward aryl α-mannopyranosides. To circumvent this problem, we describe a convenient synthesis of the fluorogenic disaccharide substrate 4-methylumbelliferone α-d-mannopyranosyl-(1→6)-β-d-mannopyranoside. We show this substrate can be used in a coupled fluorescent assay by using β-mannosidases from either Cellulomonas fimi or Helix pomatia as the coupling enzyme. We find that this disaccharide substrate is processed much more efficiently than aryl α-mannopyranosides by CpGH125, most likely because inclusion of the second mannose residue makes this substrate more like the natural host glycan substrates of this enzyme, which enables it to bind better. Using this sensitive coupled assay, the detailed characterization of these metal-independent exo-α-mannosidases GH125 enzymes should be possible, as should screening chemical libraries for inhibitors of these virulence factors.     

Mechanistic studies on AMD6221: a ruthenium-based nitric oxide scavenger

Nitric oxide is a mediator of many disease states. Previous studies have demonstrated that ruthenium(III) polyaminocarboxylates can react with NO to form stable complexes reducing the levels of nitrite in the culture medium of stimulated RAW264 macrophages and reverse the NO-mediated hypotension in animal models of septic shock. It was necessary to confirm that these observations were due to NO scavenging and not inhibition of the NO metabolic pathway. Using RAW264 cells it was confirmed that [Ru(H(3)dtpa)(Cl)] (AMD6221) was neither acting at the level of iNOS induction, nor as an inhibitor of iNOS by measuring iNOS mRNA by RT-PCR and protein by Western blot and enzyme activity. Using HPLC, the nitrosyl adduct of reaction of AMD6221, [Ru(H(2)dtpa)NO], was identified in the medium of stimulated RAW264 cells co-incubated with AMD6221, concomitant with a stoichiometric reduction in nitrite/nitrate levels, thus confirming that the ruthenium(III) polyaminocarboxylates exert their pharmacological effect by scavenging NO.     

Interaction of Mycobacterium ulcerans with Mosquito Species: Implications for Transmission and Trophic Relationships

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a severe necrotizing skin disease that causes significant morbidity in Africa and Australia. Person-to-person transmission of Buruli ulcer is rare. Throughout Africa and Australia infection is associated with residence near slow-moving or stagnant water bodies. Although M. ulcerans DNA has been detected in over 30 taxa of invertebrates, fish, water filtrate, and plant materials and one environmental isolate cultured from a water strider (Gerridae), the invertebrate taxa identified are not adapted to feed on humans, and the mode of transmission for Buruli ulcer remains an enigma. Recent epidemiological reports from Australia describing the presence of M. ulcerans DNA in adult mosquitoes have led to the hypothesis that mosquitoes play an important role in the transmission of M. ulcerans. In this study we have investigated the potential of mosquitoes to serve as biological or mechanical vectors or as environmental reservoirs for M. ulcerans. Here we show that Aedes aegypti, A. albopictus, Ochlerotatus triseriatus, and Culex restuans larvae readily ingest wild-type M. ulcerans, isogenic toxin-negative mutants, and Mycobacterium marinum isolates and remain infected throughout larval development. However, the infections are not carried over into the pupae or adult mosquitoes, suggesting an unlikely role for mosquitoes as biological vectors. By following M. ulcerans through a food chain consisting of primary (mosquito larvae), secondary (predatory mosquito larva from Toxorhynchites rutilus septentrionalis), and tertiary (Belostoma species) consumers, we have shown that M. ulcerans can be productively maintained in an aquatic food web.Infection with Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU) disease, is associated with residence near stagnant and slow-moving water bodies in areas in which the disease is endemic (5, 36, 40, 45, 50). A plasmid-encoded macrolide toxin, mycolactone, is the primary virulence determinant of M. ulcerans (8, 41). Biting aquatic insects, such as several taxa in the Belostomatidae and Naucoridae families (Hemiptera), have been suggested as possible vectors of M. ulcerans in several laboratory experiments (16, 19, 20, 24, 31, 32); however, there is little empirical evidence from field studies to support the contention that these biting insects vector M. ulcerans to humans (2). In Melbourne, Australia, recent epidemiological evidence suggests that mosquitoes may serve as vectors in the transmission of BU disease (10, 11, 12, 34, 35). In this study, 957 pools consisting of over 11,000 mosquitoes of four different species were collected and tested by quantitative PCR (qPCR) for the presence of M. ulcerans DNA, and positive results were obtained from 48 of 957 pools tested (10). Of the 48 positive pools, 13 were positive for PCR directed against two insertion sequences (IS2404 and IS2606) as well as against sequence based on the ketoreductase domain of the mycolactone toxin genes. Because all of these target sequences are present multiple times in the genome, it was difficult to assign genome equivalents to these results. However, data from laboratory experiments suggested that 10 to 100 M. ulcerans isolates per mosquito were present in the positive pools. Epidemiological work also suggested a seasonal relationship between Buruli ulcer and mosquito-vectored diseases in Australia (12). These studies are extremely provocative and raise a number of questions for further work. What is the prevalence of M. ulcerans in other invertebrate taxa in the same environment? What is the infection rate in equal numbers of mosquitoes collected from areas in which the disease is not endemic? Is it possible to obtain physical evidence for the presence of M. ulcerans through microscopy or culture of mosquitoes in areas in which the disease is endemic, and, finally, what can we learn from laboratory studies concerning the interaction between mosquitoes and M. ulcerans?The recent work from Australia suggesting that M. ulcerans is spread by mosquitoes is particularly significant because adult mosquitoes are the most important group of insects in the spread of human disease. They may serve as biological vectors that provide a major environment for pathogen replication, as in malaria or yellow fever, or as mechanical vectors that carry organisms between hosts without serving as a site of replication (1, 4, 7, 9, 38). Larval mosquitoes are common in habitats associated with BU disease, most notably lentic or standing water habitats, and feed by filtering particles in the water using labral head fans (21). Members of some genera (i.e., Anopheles) aggregate at the air-water interface in microlayers near plant stems and algal mats (27, 28, 46), where they feed on microorganisms such as bacteria and algae (47). Because of their collecting-filtering feeding mode, there is potential for larvae to consume M. ulcerans and concentrate mycobacteria through their feeding activities (22, 23).In Ghana, the occurrence of M. ulcerans among invertebrate communities in lentic habitats has been documented from regions in Ga West and Ga East Districts in which the disease is endemic as well as those in which it is not endemic (2, 49) but not in geographically distinct areas in which the disease is not endemic such as the Volta region (49). M. ulcerans has been identified in a suite of environmental samples such as filtered water, biofilms, and algae as well as among a broad spectrum of invertebrate taxa, including both larval and adult mosquitoes (2, 11, 17, 49). However, the replication and trophic movement of M. ulcerans within these environmental samples and invertebrate communities have not been experimentally investigated. Conceptual models have been proposed that assume that the primary consumers of M. ulcerans (e.g., mosquito larvae, cladocerans, and chironomid larvae) may feed on bacteria and algae in biofilms, filter suspended matter from the water column, and then initiate the passage of M. ulcerans through an aquatic food web (2, 22, 31). This model predicts the movement of M. ulcerans through secondary and tertiary consumers and implies a complex trophic relationship in the ecology of M. ulcerans as well as an important role of aquatic invertebrates in the disease ecology of M. ulcerans.In the studies reported here, we have explored the role of mosquitoes as biological or mechanical vectors of M. ulcerans, as well as the potential of mosquito larvae to play a central role in the movement of M. ulcerans through an aquatic food web. In order to investigate the ability of mosquito larvae to ingest and maintain M. ulcerans within their digestive tract as well as to persist throughout the mosquito development cycle, we took advantage of the fact that mosquito larvae naturally feed upon bacteria. Results presented here show that strains of M. ulcerans from Africa and Australia, as well as Mycobacterium marinum, were maintained at high levels in the larval mosquito gut for 6 days. However, neither M. ulcerans nor M. marinum was detected in adult mosquitoes that were infected in the larval stage. These results suggest that mosquitoes are unlikely to serve as biological vectors of M. ulcerans.We further developed a model for following the passage of M. ulcerans through a series of consumers to determine whether M. ulcerans could be passed up a trophic chain from primary to tertiary consumers. In this model, we conducted similar experiments using four species of nonpredatory mosquito larvae, Aedes aegypti (Linnaeus), Aedes albopictus (Skuse), Ochlerotatus triseriatus (Theobald), and Culex restuans (Theobald), as primary consumers. These larvae were infected with isogenic wild-type (WT) and toxin-negative isolates of M. ulcerans and of M. marinum, the closest relative to M. ulcerans (13, 14, 51). We have shown that M. ulcerans in mosquito larvae survive passage through secondary and tertiary consumers, thus providing the first laboratory evidence that M. ulcerans has the potential to move between and be maintained within different species in an aquatic food web.     

Methyl Jasmonate Increases the Tropane Alkaloid Scopolamine and Reduces Natural Herbivory in Brugmansia suaveolens: Is Scopolamine Responsible for Plant Resistance?

The tropane alkaloid (TA) scopolamine is suggested to protect Brugmansia suaveolens (Solanaceae) against herbivorous insects. To test this prediction in a natural environment, scopolamine was induced by methyl jasmonate (MJ) in potted plants which were left 10?days in the field. MJ-treated plants increased their scopolamine concentration in leaves and herbivory decreased. These findings suggest a cause?Ceffect relationship. However, experiments in laboratory showed that scopolamine affect differently the performance of the specialist larvae of the ithomiine butterfly Placidina euryanassa (C. Felder & R. Felder) and the generalist fall armyworm Spodoptera frugiperda (J. E. Smith): the specialist that sequester this TA from B. suaveolens leaves was not negatively affected, but the generalist was. Therefore, scopolamine probably acts only against insects that are not adapted to TAs. Other compounds that are MJ elicited may also play a role in plant resistance against herbivory by generalist and specialist insects, and deserve future investigations.     

Cyanotoxins in small artificial dams in Kenya utilised for cage fish farming – a threat to local people?

Nine small artificial dams located in different climatic regions of Kenya were studied. The local communities use the stored water for various purposes, such as irrigation, domestic use, watering of livestock and cage fish farming. Such intense use is commonly accompanied by eutrophication, including fast growth of cyanobacteria, which at times produce cyanotoxins threatening human and animal life. We studied the pelagic community, analysed abiotic variables and identified microcystins by means of high performance liquid chromatography and ELISA kits at monthly intervals over a period of one year. Mass spectrometry (MALDI-TOF MS) was used to identify structural variants of microcystins by their protonated masses (M + H). Three dams contained microcystins, with the highly toxic Microcystin-LR being identified as the most prominent substance. Cell content of the toxin varied from 7.2 to 686.7 fg cell^?1. Basic limnological variables that indicate the probability of toxin presence were also recorded. Non-parametric Mann–Whitney U-test revealed significant differences in soluble reactive phosphorous, nitrate-N, water depth, total hardness and post-Nauplii stages sampled between toxin-producing and non-toxin-producing dams. Although most of the samples did not contain high amounts of cyanobacteria, the cyanotoxin-problem was evident, suggesting the need for regular cyanotoxin monitoring programs.     

Mycobacterium ulcerans Fails to Infect through Skin Abrasions in a Guinea Pig Infection Model: Implications for Transmission

Transmission of M. ulcerans, the etiological agent of Buruli ulcer, from the environment to humans remains an enigma despite decades of research. Major transmission hypotheses propose 1) that M. ulcerans is acquired through an insect bite or 2) that bacteria enter an existing wound through exposure to a contaminated environment. In studies reported here, a guinea pig infection model was developed to determine whether Buruli ulcer could be produced through passive inoculation of M. ulcerans onto a superficial abrasion. The choice of an abrasion model was based on the fact that most bacterial pathogens infecting the skin are able to infect an open lesion, and that abrasions are extremely common in children. Our studies show that after a 90d infection period, an ulcer was present at intra-dermal injection sites of all seven animals infected, whereas topical application of M. ulcerans failed to establish an infection. Mycobacterium ulcerans was cultured from all injection sites whereas infected abrasion sites healed and were culture negative. A 14d experiment was conducted to determine how long organisms persisted after inoculation. Mycobacterium ulcerans was isolated from abrasions at one hour and 24 hours post infection, but cultures from later time points were negative. Abrasion sites were qPCR positive up to seven days post infection, but negative at later timepoints. In contrast, M. ulcerans DNA was detected at intra-dermal injection sites throughout the study. M. ulcerans was cultured from injection sites at each time point. These results suggest that injection of M. ulcerans into the skin greatly facilitates infection and lends support for the role of an invertebrate vector or other route of entry such as a puncture wound or deep laceration where bacteria would be contained within the lesion. Infection through passive inoculation into an existing abrasion appears a less likely route of entry.     

Aflatoxicosis in rabbits: Effectiveness of Egyptian raw bentonite in prevention or diminution the detrimental effects of naturally aflatoxin contaminated diets

Thirty five females and 15 males of New Zealand White mature rabbits about 6 months of age, were assigned to 1–5 dietary treatments (7 does+3 bucks for each): uncontaminated control diet, naturally aflatoxin contaminated diet without or with 1,2 and 3% bentonite. Rabbit fed with the aflatoxin-diet had a decreased (P<0.01 or 0.05) physical semen characteristics of bucks and a reproductive performance traits of does. The values of conception rate (%), gestation length (days), litter size (n) and litter weights (g) at birth and viability (%) of litters of doe rabbits, fed with the aflatoxin-diet, recorded, respectively: 64.5; 31.0; 4.4; 275.0 and 57.1 versus 85.6; 30.3; 7.9; 508.0; and 100 for those fed with the uncontaminated diet. Addition of bentonite to the aflatoxin contaminated diet improved in general the physical semen characteristics of buck and reproductive performance traits of doe rabbits. The results of the study demonstrate that adding 1% of Egyptian raw bentonite to the naturally aflatoxin contaminated rabbit diets can provide an effective, cheap and safe practical technique for preventing the aflatoxicosis in mature rabbits.