Bioactivity guided isolation of antifungal compounds from the liverwort Bazzania trilobata (L.) S.F. Gray

A dichloromethane and a methanol extract of the liverwort Bazzania trilobata showed antifungal activity against the phytopathogenic fungi Botrytis cinerea, Cladosporium cucumerinum, Phythophthora infestans, Pyricularia oryzae and Septoria tritici. Bioautography on thin-layer chromatograms was used to isolate six antifungal sesquiterpenes: 5- and 7-hydroxycalamenene, drimenol, drimenal, viridiflorol, gymnomitrol and three bisbibenzyls: 6 ',8'-dichloroisoplagiochin C, isoplagiochin D and 6'-chloroisoplagiochin D. Furthermore we report the isolation of gymnomitr-8(12)-en-4-one and the new coumarin 7,8-dihydroxycoumarin-7-O-beta-D-glucuronide. Their structures have been elucidated based on extensive NMR spectral evidence.     

Lignan derivatives from the liverwort Bazzania trilobata

Eight lignan derivatives trilobatin D-K, as well as jamesopyrone were isolated from the liverwort Bazzania trilobata. Their structures have been elucidated based on extensive NMR spectral evidence.     

Fluorophenol oxidation by a fungal chloroperoxidase

Caldariomyces fumago chloroperoxidase degrades monofluorophenols at both pH 3 and pH 6. 4-Fluorophenol is most readily degraded and its oxidation is most efficient at pH 6. GC-MS analyses of the reaction products revealed compounds relating to the reaction of fluorophenol radical. The degradation of fluorinated compounds is of significant environmental interest and this versatile enzyme may by employed to treat contaminated soil or water prior to discharge.     

First detection of Nosema ceranae, a microsporidian parasite of European honey bees (Apis mellifera), in Canada and central USA

Nosema ceranae is an emerging microsporidian parasite of European honey bees, Apis mellifera, but its distribution is not well known. Six Nosema-positive samples (determined from light microscopy of spores) of adult worker bees from Canada (two each from Nova Scotia, New Brunswick, and Prince Edward Island) and two from USA (Minnesota) were tested to determine Nosema species using previously-developed PCR primers of the 16S rRNA gene. We detected for the first time N. ceranae in Canada and central USA. One haplotype of N. ceranae was identified; its virulence may differ from that of other haplotypes.     

First detection of the microsporidium Enterocytozoon bieneusi in non-mammalian hosts (chickens)

Faecal samples taken from eight underweight, approximately 5-week-old broiler chickens in a poultry abattoir were investigated for microsporidial infections by light microscopy, electron microscopy, and PCR. In two of six chickens, which were suspected of being infected with microsporidia by light microscopy, Enterocytozoon bieneusi (genotype 'J') was detected by PCR and DNA sequencing, and in one of the two PCR-positive samples by extensive electron microscopy. This is the first time that E. bieneusi has been detected in chickens, i.e. in a non-mammalian species.     

Vascular Streak Dieback of cacao in Southeast Asia and Melanesia: in planta detection of the pathogen and a new taxonomy

Vascular Streak Dieback (VSD) disease of cacao (Theobroma cacao) in Southeast Asia and Melanesia is caused by a basidiomycete (Ceratobasidiales) fungus Oncobasidium theobromae (syn. =Thanatephorus theobromae). The most characteristic symptoms of the disease are green-spotted leaf chlorosis or, commonly since about 2004, necrotic blotches, followed by senescence of leaves beginning on the second or third flush behind the shoot apex, and blackening of infected xylem in the vascular traces at the leaf scars resulting from the abscission of infected leaves. Eventually the shoot apex is killed and infected branches die. In susceptible cacao the fungus may grow through the xylem down into the main stem and kill a mature cacao tree. Infections in the stem of young plants prior to the formation of the first 3-4 lateral branches usually kill the plant. Basidiospores released from corticioid basidiomata developed on leaf scars or along cracks in the main vein of infected leaves infect young leaves. The pathogen commonly infects cacao but there are rare reports from avocado. As both crops are introduced to the region, the pathogen is suspected to occur asymptomatically in native vegetation. The pathogen is readily isolated but cultures cannot be maintained. In this study, DNA was extracted from pure cultures of O. theobromae obtained from infected cacao plants sampled from Indonesia. The internal transcribed spacer region (ITS), consisting of ITS1, 5.8S ribosomal RNA and ITS2, and a portion of nuclear large subunit (LSU) were sequenced. Phylogenetic analysis of ITS sequences placed O. theobromae sister to Ceratobasidium anastomosis groups AG-A, AG-Bo, and AG-K with high posterior probability. Therefore the new combination Ceratobasidium theobromae is proposed. A PCR-based protocol was developed to detect and identify C. theobromae in plant tissue of cacao enabling early detection of the pathogen in plants. A second species of Ceratobasidium, Ceratobasidium ramicola, identified through ITS sequence analysis, was isolated from VSD-affected cacao plants in Java, and is widespread in diseased cacao collected from Indonesia.     

Nocardiosis in Mediterranean bivalves: First detection of Nocardia crassostreae in a new host Mytilus galloprovincialis and in Ostrea edulis from the Gulf of Naples (Italy)

In this work M. galloprovincialis and O. edulis specimens were surveyed for a pathological study in the Gulf of Naples (Mediterranean sea, Campania Region, southern Italy). Clusters of Nocardia sp.-like cells were observed in histological slides. PCR amplification, sequencing and in situ hybridization were carried out in order to corroborate Nocardia species identification for both hosts. Blast results showed a 99% of maximum identity with Nocardia crassostreae sequences in Genbank. This is the first report of N. crassostreae in the new host M. galloprovincialis and, in a new area, the Mediterranean Sea.     

Role of H164 in a unique dye-decolorizing heme peroxidase DyP

The expression system of a unique dye-decolorizing peroxidase DyP in Escherichia coli has been constructed. The molecular mass of the expressed DyP (eDyP) is 47kDa, indicating no any modification with saccharides. The characteristics of eDyP were almost the same as those of native DyP from a fungus Thanatephorus cucumeris Dec 1 and recombinant DyP with Aspergillus oryzae except thermostability. As H164 was suggested to be the proximal histidine based on the preliminary X-ray crystallographic analysis of DyP, the site-directed mutations H164A and H166A (residue near H164) were introduced into the gene encoding DyP. The specific activity and RZ value of the purified H164A were 1.52U/mg and 0.11, respectively, which were 99.8% and 95% lower than those of eDyP, respectively. On the contrary, those of H166A were not different from those of eDyP. Therefore, H164 was confirmed to be the proximal histidine.     

Akirins in sea lice: First steps towards a deeper understanding

Sea lice (Copepoda, Caligidae) are the most widely distributed marine pathogens in the salmon industry. Vaccination could be an environmentally friendly alternative for sea lice control; however, research on the development of such vaccines is still at an early stage of development. Recent results have suggested that subolesin/akirin/my32 are good candidate antigens for the control of arthropod infestations, including sea lice, but background knowledge about these genes in crustaceans is limited. Herein, we characterize the my32 gene/protein from two important sea lice species, Caligus rogercresseyi and Lepeophtheirus salmonis, based on cDNA sequence isolation, phylogenetic relationships, three dimensional structure prediction and expression analysis. The results show that these genes/proteins have the main characteristics of akirins from invertebrates. In addition, immunization with purified recombinant my32 from L. salmonis elicited a specific antibody response in mice and fish. These results provide an improvement to our current knowledge about my32 proteins and their potential use as vaccine candidates against sea lice in fish.     

Sesquiterpene constituents from the essential oils of the liverworts Mylia taylorii and Mylia nuda

The essential oils and extracts of Mylia taylorii and M. nuda were investigated by gas chromatography, mass spectrometry, NMR spectroscopy and chemical correlations. Beside several known compounds 13 new constituents including three new carbon skeletons could be identified. Four hydrocarbons with a molecular formula of C₁₅H₂₂ (m/z 202) were identified as myli-4(15)-ene (1), aromadendra-1(10),4(15)-diene (19), aromadendra-4,10(14)-diene (20) and aromadendra-4,9-diene (21). Three oxaspiro-compounds were identified as 7-epi-bourbon-3-en-5,11-oxide (22), guai-3,10(14)-dien-5,11-oxide (23) and guai-3,9-dien-5,11-oxide (24). The absolute configuration of myli-4(15)-en-3-one (5) could be established by chemical correlation. Together with α-taylorione (7) the corresponding 6,11-seco-compound taylopyran (25) with a new carbon skeleton was identified which serves as a precursor to taylocyclane (26) and taylofuran (27). Taynudol (28) contains a new carbon skeleton with a cyclobutenyl structure.     

Sesquiterpene constituents from the liverwort Bazzania japonica

The hydrodistillation products of the liverwort Bazzania japonica were separated by preparative gas chromatography (GC) and investigated by spectroscopic methods. Seven unknown compounds were isolated and identified by GC-MS and NMR. Four of them, the norsesquiterpene hydrocarbons 4-epi-11-nor-aristola-1(10),11-diene (1), 4-epi-11-nor-aristola-1,9,11-triene (2), 4-epi-11-nor-aristola-9,11-diene (3), and one oxygenated sesquiterpene, (-)-aristol-1(10)-en-12-ol (5) are new natural compounds, and one, (+)-himachala-2,4-diene (7), has for the first time been isolated from liverworts. The absolute configurations of 5 and 7 were derived by chemical correlation reactions and/or enantioselective GC using cyclodextrin phases. 1, 2 and 3 have identical absolute configuration.     

Development of a real-time SYBRGreen PCR assay for rapid detection of acquired AmpC in Enterobacteriaceae

Introduction

Acquired AmpC enzymes, classified as miscellaneous extended-spectrum β-lactamase (ESBL_M) enzymes according to a recently proposed β-lactamase classification, are increasing according to several publications. Simple and rapid methods for detection of ESBL_M are needed for appropriate infection control. A gel-based multiplex PCR method for acquired bla_AmpC detection and subtype classification has been available for several years. Here, we describe a modification of the protocol to suit real-time PCR platforms and to include novel genotypes.

Material and methods

Clinical isolates with clavulanic acid non-reversible non-susceptibility to extended-spectrum cephalosporins were subjected to combination disk testing with cefoxitin +/− cloxacillin at Malmö University Hospital. Phenotypical AmpC production was defined as cloxacillin reversible cefoxitin resistance. In this study 51 phenotypical AmpC-producing isolates, were subjected to the acquired bla_AmpC real-time PCR assay. The acquired blaAmpC positive isolates were further characterized by DNA sequencing of the acquired AmpC encoding gene, Pulsed-Field Gel Electrophoresis (PFGE) and PCR-based replicon typing.

Results and discussion

The real-time PCR assay was able to detect and sub-classify all acquired bla_AmpC genes described to date. The assay can be performed in less than 3 h, including pre-PCR preparations. Analysis of the isolate collection resulted in 18 of 51 phenotypical AmpC-producing isolates being positive in the acquired bla_AmpC real-time multiplex PCR assay; 17 of subtype CIT and one DHA. Sequence analysis identified 16 isolates as blaCMY-2, one as blaCMY-16 and one as blaDHA-1. Detected plasmid replicon types were I1 and B/O. Two of the E. coli isolates were identical according to PFGE and the others were unrelated.     

First in-gel detection and purification of human xylosyltransferase II

Human xylosyltransferases I and II (XylT-I, XylT-II) are key enzymes in glycosaminoglycan biosynthesis. Knowledge about the in vivo molecular weight, oligomeric state or turnover number are essential characteristics which have been addressed in this study. XylT-II was purified from Pichia pastoris by fractionated ammonium sulfate precipitation, heparin affinity and ion exchange chromatography. XylT-II was purified over 7000-fold with a final yield of 2.6%. By utilizing mass spectra analysis we can prove its first in-gel detection showing a migration pattern behavior that confirms its in silico molecular weight of 95.8 kDa. We could determine a turnover number of 2.18 min⁻¹ or one transferred xylose molecule per one XylT-II molecule each 27.5 s. The k_cat/K_M ratio was 0.357 min⁻¹ μM⁻¹ for XylT-II using the bikunin-homologous acceptor Bio-QEEEGSGGGQKK-F. The comparison to XylT-I derived from the same organism revealed a 2.4-fold higher catalytic efficiency (0.870 min⁻¹ μM⁻¹) for XylT-I.     

First record of a nematode Metastrongyloidea (Aelurostrongylus abstrusus larvae) in Achatina (Lissachatina) fulica (Mollusca, Achatinidae) in Brazil

Achatina (Lissachatina) fulica was introduced in Brazil in the 1980s for commercial purposes (“escargot” farming) and nowadays, mainly by human activity, it is widespread in at least 23 out of 26 Brazilian states and Brasília, including the Amazonian region and natural reserves, where besides a general nuisance for people it is a pest and also a public health concern, since it is one of the natural intermediate host of Angiostrongylus cantonensis, ethiological agent of the meningoencephalitis in Asia. As Brazil is experiencing the explosive phase of the invasion, the Laboratório de Malacologia do Instituto Oswaldo Cruz/Fiocruz has been receiving samples of these molluscs for identification and search for Angiostrongylus cantonensis and Angiostrongylus costaricensis larvae. While examining samples of A. fulica different nematode larvae were obtained, including Aelurostrongylus, whose different species are parasites of felids, dogs, primates, and badger. Morphological and morphometric analyses presented herein indicated the species Aelurostrongylus abstrusus, as well as the occurrence of other nematode larvae (Strongyluris-like) found in the interior of the pallial cavity of A. fulica. This is the first report in Brazil of the development of A. abstrusus infective larvae in A. fulica evidencing the veterinary importance of this mollusc in the transmission of A. abstrusus to domestic cats. Since the spread of A. fulica is pointed out in the literature as one of the main causative spread of the meningoencephalitis caused by A. cantonensis the authors emphasize the need of sanitary vigilance of snails and rats from vulnerable areas for A. cantonensis introduction as the port side areas.     

First report of a mixed infection due to Acanthamoeba genotype T3 and Vahlkampfia in a cosmetic soft contact lens wearer in Iran

Acanthamoeba keratitis cases have emerged in the recent years in Iran. In this case, an amoebic keratitis due to a mixed infection with Acanthamoeba and Vahlkampfia species is reported. Corneal scrapes, contact lenses and contact lens cases obtained from the patient were analysed and were positive for cysts of Acanthamoeba and Vahlkampfia genera. Genus-specific PCR was carried out for both genera, confirming the microscopic observations. To our knowledge, this is the first reported case of a possible mixed amoebic infection due to Acanthamoeba and Vahlkampfia and raises awareness within contact lens wearers in Iran.     

Chemical constituents of Malagasy liverworts: cyclomyltaylanoids from Bazzania madagassa

Five cyclomyltaylanoids (2-6), together with 1R,5R-diacetoxycyclomyltaylan-10-one (1), (+)-globulol, and ent-4beta,10alpha-dihydroxyaromadendrane have been isolated from the diethyl ether fraction of the Malagasy liverwort, Bazzania madagassa. The structure of 1 was confirmed by X-ray analysis, while those of the compounds were established on the basis of one- and two-dimensional NMR spectroscopic evidence, and comparison with data reported in the literature. The chemosystematics of B. madagassa are discussed.     

First report of Pandora neoaphidis resting spore formation in vivo in aphid hosts

The entomopathogenic fungus Pandora neoaphidis is a recognized pathogen of aphids, causes natural epizootics in aphid populations, and interacts and competes with aphid predators and parasitoids. Survival of entomophthoralean fungi in periods of unsuitable weather conditions or lack of appropriate host insects is accomplished mainly by thick-walled resting spores (zygospores or azygospores). However, resting spores are not known for some entomophthoralean species such as P. neoaphidis. Several hypotheses of P. neoaphidis winter survival can be found in the literature but so far these hypotheses do not include the presence of resting spores. Resting spores were found in an aphid population where P. neoaphidis was the only entomophthoralean fungus observed during surveys conducted in organic horticultural crops in greenhouses and open fields in Buenos Aires province, Argentina. This study sought to use molecular methods to confirm that these resting spores were, in fact, those of P. neoaphidis while further documenting and characterizing these resting spores that were produced in vivo in aphid hosts. The double-walled resting spores were characterized using light and transmission electron microscopy. The Argentinean resting spores clustered together with P. neoaphidis isolates with bootstrap values above 98 % in the small subunit ribosomal RNA (SSU rRNA) sequence analysis and with bootstrap values above 99 % the Internal Transcribed Spacer (ITS) II region sequence analysis. This study is the first gene-based confirmation from either infected hosts or cultures that P. neoaphidis is able to produce resting spores.     

First description of the disease by Conidiobolus osmodes on Tipula paludosa larvae with the report of a natural epizootic

A new fungal pathogen of Tipula paludosa (Tipulidae: Diptera) larvae, Conidiobolus osmodes (Ancylistaceae: Entomophthorales), was found during a survey of tipulid larval pathogens in Northumbria and Cumbria in England in 1997-1999. The fungus caused an epizootic in a population at Close House during autumn 1999 and spring 2000 with prevalence rising fourfold reaching about 40% in April 2000. The disease development was presented and the fungus was described from naturally infected larvae and artificial cultures.     

First record of Pseudomyicola spinosus in Argopecten ventricosus in Baja California, Mexico

This is the first record of the copepod Pseudomyicola spinosus in the scallop Argopecten ventricosus in northwestern Mexico, and describes: (1) the known annual prevalence and intensity of this copepod on scallops from culture sites (Gulf of California) and natural populations (Pacific coast), (2) the histopathological effects caused on the soft tissues of scallops, and (3) the relationship between prevalence and intensity records and environmental parameters. The copepod was present throughout the period of investigation, showing similar prevalence and ratio of copepod to scallop patterns in both cultured scallops and wild specimens from natural habitats. Highest prevalence and ratio values were detected in summer-autumn at both sites, probably because scallops showed a weak condition from the combined effects of spawning, reabsorption of residual gametes, and high temperature. The condition index of A. ventricosus showed a significant correlation with the presence of the copepod in Magdalena Bay (-0.67). P. spinosus was observed in the gills of scallops, producing alterations or rupture of filaments, and in the stomach, causing detachment and loss of the epithelium. No relationship between copepod infestation with temperature, salinity, chlorophyll, and seston were found during the investigation. Although P. spinosus was present year-round at both sites, no association between infestation and scallop mortalities was detected.