Evaluation of real-time PCR targeting hbb gene for Borrelia species identification

Several molecular methods have been employed for Borrelia species identification. Newly developed technology, real-time polymerase chain reaction (RT-PCR), combines simultaneous amplification, detection and differentiation of strains in one PCR run. The aim of the study was to perform and evaluate RT-PCR for Borreliaburgdorferi sensu lato species identification. Borrelia species identification was accomplished on 374 Borrelia strains using two approaches: 1.) MluI restriction of entire borrelial chromosome (MluI-large restriction fragment patterns, LRFP), and 2.) RT-PCR targeting hbb gene and specific melting temperature (Tm) detection. The results of the two molecular methods were compared. With MluI-RFLP we were able to differentiate all Borrelia species and their subtypes within particular species. RT-PCR based on Tm determination identified unique strains within the species Borreliaafzelii (Tm 66.11 °C), B. burgdorferi sensu stricto (Tm 68.18 °C), Borreliaspielmanii (Tm 59.45 °C) and Borreliavalaisiana (Tm 59.62 °C). We were not able to distinguish the last two species that shared almost identical Tm. The large majority of Borreliagarinii strains shared Tm 51.42 °C, while subtype Mlg4 was characterized by Tm 56.87 °C. Strains of Borrelialusitaniae species also were heterogeneous; human isolate had Tm 63.47 °C while two tick isolates shared Tm 61.77 °C. Differences inside hbb gene enabled differentiation of the majority of Borrelia species, and revealed two clusters within B. garinii and B. lusitaniae species, respectively, but it was not possible to distinguish B. spielmanii form B. valaisiana. The major advantage of RT-PCR was that it was easy to perform and that the results were obtained within a few hours.     

A new species of ferret-badger, Genus Melogale, from Vietnam

Ferret-badgers, genus Melogale, are distributed in the Indochinese region, Java, Bali and NE-Borneo. There are currently four species described each having very similar phenotypes. In March 2005, a living ferret-badger of a different phenotype was confiscated by rangers from Cuc Phuong National Park, Vietnam. This individual died and the carcass was not preserved. In January 2006, a newly deceased individual of the same phenotype was found at the Endangered Primate Rescue Center, Cuc Phuong National Park. Due to several different characteristics these individuals vary greatly from the current species. Thus, we describe an additional species, M. cucphuongensis sp. nov. from northern Vietnam, which occurs sympatrically with M. moschata and M. personata, but differs from both species clearly in skull morphology and other features.Based on a 423 bp-long fragment of the mitochondrial cytochrome b gene, M. cucphuongensis sp. nov. is a member of the genus Melogale and represents a sister lineage to a clade consisting of M. personata and M. moschata.     

Chemotaxonomy of Pseudowintera: Sesquiterpene dialdehyde variants are species markers

Two sesquiterpene dialdehydes, the 1β-E-coumaroyl-5α-hydroxypolygodial plus the known 1β-E-cinnamoyl-5α-hydroxypolygodial, were isolated from the recently described species Pseudowintera insperata. This discovery is a further example of the rare sesquiterpene dialdehyde coumarate/cinnamate combination being found exclusively in the family Winteraceae. Another sesquiterpene dialdehyde, isopaxidal, with the rare rearranged drimane skeleton, was isolated from Pseudowintera axillaris. The sesquiterpene dialdehyde contents of leaves of 25 individual plants of the four Pseudowintera species, all endemic to New Zealand, were measured by HPLC. P. insperata individuals all had high levels (3.0-6.9% of leaf dry wt.) of the coumarate, P. axillaris had high levels (2.2-6.9%) of paxidal, and Pseudowintera colorata from different areas of New Zealand contained varying levels of polygodial (1.4-2.9%) and 9-deoxymuzigadial (0-2.9%). Therefore the sesquiterpene dialdehydes are good species markers.     

Virulence factors are released in association with outer membrane vesicles of Pseudomonas syringae pv. tomato T1 during normal growth

Outer membrane vesicles (OMVs) are released from Pseudomonas syringae pv. tomato T1 (Pst T1) during their normal growth. These extracellular compartments are comprised of a complete set of biological macromolecules that includes proteins, lipids, lipopolysaccharides, etc. It is evident from proteomics analyses the OMVs of Pst T1 contain membrane- and virulence-associated proteins. In addition, OMVs of this organism are also associated with phytotoxin, coronatine. Therefore, OMVs of Pst T1 must play a significant role during pathogenicity to host plant. However, further studies are required whether these structures can serve as “vehicles” for the transport of virulence factors into the host membrane.     

Wild peanut Arachis duranensis are nodulated by diverse and novel Bradyrhizobium species in acid soils

Aiming at learning the microsymbionts of Arachis duranensis, a diploid ancestor of cultivated peanut, genetic and symbiotic characterization of 32 isolates from root nodules of this plant grown in its new habitat Guangzhou was performed. Based upon the phylogeny of 16S rRNA, atpD and recA genes, diverse bacteria belonging to Bradyrhizobium yuanmingense, Bradyrhizobium elkanii, Bradyrhizobium iriomotense and four new lineages of Bradyrhizobium (19 isolates), Rhizobium/Agrobacterium (9 isolates), Herbaspirillum (2 isolates) and Burkholderia (2 isolates) were defined. In the nodulation test on peanut, only the bradyrhizobial strains were able to induce effective nodules. Phylogeny of nodC divided the Bradyrhizobium isolates into four lineages corresponding to the grouping results in phylogenetic analysis of housekeeping genes, suggesting that this symbiosis gene was mainly maintained by vertical gene transfer. These results demonstrate that A. duranensis is a promiscuous host preferred the Bradyrhizobium species with different symbiotic gene background as microsymbionts, and that it might have selected some native rhizobia, especially the novel lineages Bradyrhizobium sp. I and sp. II, in its new habitat Guangzhou. These findings formed a basis for further study on adaptation and evolution of symbiosis between the introduced legumes and the indigenous rhizobia.     

Morphometric analysis of six Gerbillus species (Rodentia,Gerbillinae) from Tunisia

Size and shape changes in the skull of the genus Gerbillus were investigated using geometric morphometrics. Six species from Tunisia were studied (G. gerbillus, G. campestris, G. nanus, G. tarabuli, G. simoni and G. latastei). Statistical analyses of shape variability allowed us to discriminate three morphological groups which are congruent with the three groups suggested by previous morphological and molecular studies. However, our results contrast with previous molecular investigations. In fact, according to results obtained by the use of principal component analysis, canonical variate analysis and UPGMA, we found a higher degree of divergence between the subgenus Dipodillus and the other two subgenera Gerbillus and Hendecapleura. This fact suggests that the morphometric differences observed among species within the genus Gerbillus are not mainly related to phylogeny. To reconciliate the molecular and morphological approaches, we propose a hypothesis of differential rates of phenotypic evolution in the genus Gerbillus. In this view, the species belonging to the subgenus Dipodillus evolved apomorphic features of the skull likely related to a higher degree of habitat specialization. By contrast, the more generalist Gerbillus and Hendecapleura subgenera show less differentiated plesiomorphic morphology.     

Molecular differentiation and phylogenetic relationships of three Angiostrongylus species and Angiostrongylus cantonensis geographical isolates based on a 66-kDa protein gene of A. cantonensis (Nematoda: Angiostrongylidae)

The phylogenetic relationships and molecular differentiation of three species of angiostrongylid nematodes (Angiostrongylus cantonensis, Angiostrongylus costaricensis and Angiostrongylus malaysiensis) were studied using the AC primers for a 66-kDa protein gene of A. cantonensis. The AC primers successfully amplified the genomic DNA of these angiostrongylid nematodes. No amplification was detected for the DNA of Ascaris lumbricoides, Ascaris suum, Anisakis simplex, Gnathostoma spinigerum, Toxocara canis, and Trichinella spiralis. The maximum-parsimony (MP) consensus tree and the maximum-likelihood (ML) tree both showed that the Angiostrongylus taxa could be divided into two major clades - Clade 1 (A. costaricensis) and Clade 2 (A. cantonensis and A. malaysiensis) with a full support bootstrap value. A. costaricensis is the most distant taxon. A. cantonensis is a sister group to A. malaysiensis; these two taxa (species) are clearly separated. There is no clear distinction between the A. cantonensis samples from four different geographical localities (Thailand, China, Japan and Hawaii); only some of the samples are grouped ranging from no support or low support to moderate support of bootstrap values. The published nucleotide sequences of A. cantonensis adult-specific native 66 kDa protein mRNA, clone L5-400 from Taiwan (U17585) appear to be very distant from the A. cantonensis samples from Thailand, China, Japan and Hawaii, with the uncorrected p-distance values ranging from 26.87% to 29.92%.     

Antiparasitic activity of prenylated benzoic acid derivatives from Piper species

Fractionation of dichloromethane extracts from the leaves of Piper heterophyllum and P. aduncum afforded three prenylated hydroxybenzoic acids, 3-[(2E,6E,10E)-11-carboxy-3,7,15-trimethyl-2,6,10,14-hexadecatetraenyl)-4,5-dihydroxybenzoic acid, 3-[(2E,6E,10E)-11-carboxy-13-hydroxy-3,7,15-trimethyl-2,6,10,14-hexadecatetraenyl]-4,5-dihydroxybenzoic acid and 3-[(2E,6E,10E)-11-carboxy-14-hydroxy-3,7,15-trimethyl-2,6,10,15-hexadecatetraenyl]-4,5-dihydroxybenzoic acid, along with the known compounds, 4,5-dihydroxy-3-(E,E,E-11-formyl-3,7,15-trimethyl-hexadeca-2,6,10,14-tetraenyl)benzoic acid (arieianal), 3,4-dihydroxy-5-(E,E,E-3,7,11,15-tetramethyl-hexadeca-2,6,10,14-tetraenyl)benzoic acid, 4-hydroxy-3-(E,E,E-3,7,11,15-tetramethyl-hexadeca-2,6,10,14-tetraenyl)benzoic acid, 3-(3,7-dimethyl-2,6-octadienyl)-4-methoxy-benzoic acid, 4-hydroxy-3-(3,7-dimethyl-2,6-octadienyl)benzoic acid and 4-hydroxy-3-(3-methyl-1-oxo-2-butenyl)-5-(3-methyl-2-butenyl)benzoic acid. Their structures were elucidated on the basis of spectroscopic data, including homo- and heteronuclear correlation NMR experiments (COSY, HSQC and HMBC) and comparison with data reported in the literature. Riguera ester reactions and optical rotation measurements established the compounds as racemates. The antiparasitic activity of the compounds were tested against three strains of Leishmania spp., Trypanosoma cruzi and Plasmodium falciparum. The results showed that 3-(3,7-dimethyl-2,6-octadienyl)-4-methoxy-benzoic acid exhibited potent and selective activity against L. braziliensis (IC₅₀ 6.5 μg/ml), higher that pentamidine used as control. Moreover, 3-[(2E,6E,10E)-11-carboxy-3,7,15-trimethyl- 2,6,10,14-hexadecatetraenyl)-4,5-dihydroxybenzoic acid and 4-hydroxy-3-(3-methyl-1-oxo-2-butenyl)-5-(3-methyl-2-butenyl)benzoic acid showed moderate antiplasmodial (IC₅₀ 3.2 μg/ml) and trypanocidal (16.5 μg/ml) activities, respectively.     

Essential oil composition of Eryngium rosulatum P.W. Michael ined.: A new undescribed species from eastern Australia

The essential oil composition from the aerial parts of a new Eryngium species from Australia, Eryngium rosulatum P.W. Michael ined., has been analysed by GC and GC/MS. A total of 34 compounds have been identified representing around 80% of the total oil. The main constituents of the oil were found to be β-elemene (16.0%) and bicyclogermacrene (12.5%). Other representative compounds were identified as δ-elemene (7.0%) and (E)-caryophyllene (5.9%). The sesquiterpene fraction (75.0%) was predominant in the essential oil of this species, most of these were hydrocarbons (53.8%). This paper represents the first study on this new, undescribed Australian species and its chemical composition.     

Phytochemical analysis and genetic characterization of six Hypericum species from Serbia

The secondary metabolite contents and genetic profiles of six Hypericum species (H. barbatum Jacq., H. hirsutum L., H. linarioides Bosse, H. maculatum Crantz, H. rumeliacum Boiss. and H. tetrapterum Fries), collected from different locations in Serbia, have been analyzed. Methanol extracts of the aerial parts of the plants were obtained by accelerated solvent extraction (ASE) at 40 degrees C and 100 bar, and analyzed for five pharmacologically important standard constituents (hyperoside, quercitrin, pseudohypericin, hyperforin and hypericin) by LC-MS/MS. The highest content of hypericin and pseudohypericin was observed in the H. barbatum extract, while the highest content of hyperforin and quercitrin was found in the H. tetrapterum extract and the highest content of hyperoside in the H. maculatum extract. A literature survey shows that the above six Hypericum species, with the exception of H. maculatum, have not been previously genetically profiled. In order to correlate the chemical constituents of the species under investigation with their genetic factors, genetic profiling of these species was undertaken using the random amplification of polymorphic DNA (RAPD) and single sequence repeat (SSR) profiles of the above selected plants. Among the 52 random primers used for the initial screening, only 10 yielded polymorphic RAPD profiles. A total of 111 polymorphic markers were generated using these primers. The SSR analysis shows that 8 out of the 10 primers used were polymorphic. The correlation among the species under investigation using the two genetic markers was performed using Jaccuard's coefficients of similarity and a high correlation (r=0.99) was obtained. The main conclusion from the above data is that there exists a stronger correlation for secondary metabolite contents with RAPD data than with SSR data among the six Hypericum species from Serbia.     

The morphological distinction between the postcranial skeleton of Cervalces/Alces and Megaloceros giganteus and comparison between the two Alceini genera from the Upper Pliocene-Holocene of Western Europe

Alces alces, Cervalces and Megaloceros giganteus are deer of approximately the same size whose limb bones could be exchanged if fragmentary so morphological features are needed, enabling identification of postcranials remains. In this work the limb bone features described by Pfeiffer (1999a) as distinguishing Cervalces latifrons from M. giganteus are re-analyzed and further developed, considering also the vertebral column, the articular girdles and the carpals and tarsal elements. This analysis is extended to the other Cervalces species (C. gallicus, C. carnutorum and C. scotti) and to the present-day moose Alces alces. It results in a substantial uniformity in the postcranial skeleton of the species of the genus Cervalces, confirming what has already been stated by Sher (1987), that the Alceini, in the Upper Pliocene, constituted an already well-defined morphological type that did not undergo further structural postcranial remodeling.     

Essential oil composition of Hypericum L. species from Southeastern Serbia and their chemotaxonomy

The essential oils of the aerial parts of nine species of Hypericum (Hypericum barbatum, Hypericum hirsutum, Hypericum linarioides, Hypericum maculatum, Hypericum olympicum, Hypericum perforatum, Hypericum richeri, Hypericum rumeliacum and Hypericum tetrapterum), collected from different locations in Southeast Serbia, were obtained by steam distillation and analyzed by GC and GC–MS. The essential oils investigated were characterized by a high content of non-terpene compounds and a low content of monoterpenes. The contents of non-terpenes, monoterpenes and sesquiterpenes in oils of the species H. barbatum, H. richeri and H. rumeliacum (section Drosocaprium) were similar and these oils were characterized by high contents of fatty acids. The oils of H. hirsutum and H. linarioides (section Taeniocarpium) contained a high percentage of n-nonane. There were similarities in contents of non-terpenes and sesquiterpenes in oils of species that belong to the section Hypericum (H. maculatum, H. perforatum and H. tetrapterum). The oil of H. olympicum differed from others by higher terpene content. A comparison was also carried out of the chemical composition of the essential oils from flower, leaf and stem of H. perforatum and it revealed that the highest concentration of non-terpene compounds was found in the flower and stem oil, while a high concentration of sesquiterpenes was characteristic for leaf oil. There were significant differences in the concentrations of the same compounds in the essential oils of H. maculatum, H. olympicum and H. perforatum, collected in different years from the same location which could be explained by seasonal differences. All data were statistically processed with principal component analysis and cluster analysis. The main conclusion from the above data is that genetic and environmental factors both play a role in determining the composition of essential oils of the Hypericum species studied.     

Genetic relationship among Paspalum species of the subgenus Anachyris: Taxonomic and evolutionary implications

Paspalum is one of the most important genera of the Poaceae family due to its large number of species and diversity. The subgenus Anachyris comprises six species mainly from South America grouped together by sharing rare spikelet characteristics. A genetic analysis using ISSR markers, compared with the morphological and phenotypic variation observed in each one species, was used to establish genetic relationships among 40 accessions with several ploidy levels, belonging to 5 species of the subgenus Anachyris. Fourteen accessions of Paspalum malacophyllum (2x and 4x), 12 of P. simplex (2x, 3x, 4x and 6x), 4 of P. procurrens (2x and 4x), 4 of P. usterii (4x) and 6 of P. volcanensis (4x) were analysed. A total of 227 ISSR loci (98.7% polymorphic) were detected among all accessions, with variable loci number and percentages of polymorphism according to species delimitations. Six main groups were identified by cluster analysis based on Jaccard's genetic distance and UPGMA, four of which matched all the respective accessions of P. simplex, P. procurrens, P. usterii and P. volcanensis, while the other two were consistent with two different groups of accessions of P. malacophyllum, one involving most tetraploid accessions, and the other one grouping together a tetraploid and two diploid accessions. The distinctive morphological characteristics and the separate clustering of these tetraploid and diploid cytotypes suggest to consider a new multiploid species complex inside the subgenus Anachyris. Both cytotypes of P. procurrens, and the four co-specific cytotypes of P. simplex consistently clustered together forming two specific groups for the two multiploid taxons. This is in agreement with the existence of high phenotypic similarities between diploid and tetraploid cytotypes of P. procurrens, and among diploid, triploid, tetraploid and hexaploid cytotypes of P. simplex. Since the polyploid cytotypes of these species are reproduced by apomixis, the specific genetic clustering by ISSR markers and morphological and cytological results support the hypothesis that the two multiploid species were originated by autopolyploidy. Our results confirm previous studies suggesting a monophyletic origin for the subgenus Anachyris and are concordant with previous data regarding genomic homologies and phylogenetic analyses in the genus.     

Species delimitation in the European species of Clavulina (Cantharellales, Basidiomycota) inferred from phylogenetic analyses of ITS region and morphological data

The identification of the conventionally accepted species of Clavulina (Cantharellales, Basidiomycota) in Europe (Clavulina amethystina, Clavulina cinerea, Clavulina cristata, and Clavulina rugosa) is often difficult and many specimens are not straightforwardly assignable to any of those four species, which is why some authors have questioned their identity. In order to assess the status of those species, a morphological examination was combined with the molecular analysis of the ITS region. The same six major clades were obtained in the Bayesian and parsimony phylogenetic analyses, and all six clades were well-supported at least by one of the analyses. Morphological characters, such as the overall branching pattern, the presence and intensity of grey colour, the cristation of the apices, and basidiospore size and shape were to various extents correlated with the phylogenetic signal obtained from the ITS region. The congruence between the molecular analyses and morphology, rather than geographical origin, suggests the existence of several species that can be delimited using a combined phylogenetic and morphological species recognition. The analyses revealed that C. cristata and C. rugosa are well-delimited species. In contrast, more than one taxa could be subsumed under the names C. amethystina and C. cinerea, the taxonomical complexity of which is discussed. The ITS region is proved to be adequate to separate phylogenetic species of Clavulina.     

The Enterobacter aerogenes outer membrane efflux proteins TolC and EefC have different channel properties

The outer membrane proteins TolC and EefC from Enterobacter aerogenes are involved in multidrug resistance as part of two resistance-nodulation-division efflux systems. To gain more understanding in the molecular mechanism underlying drug efflux, we have undertaken an electrophysiological characterization of the channel properties of these two proteins. TolC and EefC were purified in their native trimeric form and then reconstituted in proteoliposomes for patch-clamp experiments and in planar lipid bilayers. Both proteins generated a small single channel conductance of about 80 pS in 0.5 M KCl, indicating a common gated structure. The resultant pores were stable, and no voltage-dependent openings or closures were observed. EefC has a low ionic selectivity (P_K/P_Cl = ∼ 3), whereas TolC is more selective to cations (P_K/P_Cl = ∼ 30). This may provide a possible explanation for the difference in drug selectivity between the AcrAB-TolC and EefABC efflux systems observed in vivo. The pore-forming activity of both TolC and EefC was severely inhibited by divalent cations entering from the extracellular side. Another characteristic of the TolC and EefC channels was the systematic closure induced by acidic pH. These results are discussed in respect to the physiological functions and structural models of TolC and EefC.     

Secondary metabolites from Eurotium species, Aspergillus calidoustus and A. insuetus common in Canadian homes with a review of their chemistry and biological activities

As part of studies of metabolites from fungi common in the built environment in Canadian homes, we investigated metabolites from strains of three Eurotium species, namely E. herbariorum, E. amstelodami, and E. rubrum as well as a number of isolates provisionally identified as Aspergillus ustus. The latter have been recently assigned as the new species A. insuetus and A. calidoustus. E. amstelodami produced neoechinulin A and neoechinulin B, epiheveadride, flavoglaucin, auroglaucin, and isotetrahydroauroglaucin as major metabolites. Minor metabolites included echinulin, preechinulin and neoechinulin E. E. rubrum produced all of these metabolites, but epiheveadride was detected as a minor metabolite. E. herbariorum produced cladosporin as a major metabolite, in addition to those found in E. amstelodami. This species also produced questin and neoechinulin E as minor metabolites. This is the first report of epiheveadride occurring as a natural product, and the first nonadride isolated from Eurotium species. Unlike strains from mainly infection-related samples, largely from Europe, neither ophiobolins G and H nor austins were detected in the Canadian strains of A. insuetus and A. calidoustus tested, all of which had been reported from the latter species. TMC-120 A, B, C and a sesquiterpene drimane are reported with certainty for the first time from indoor isolates, as well as two novel related methyl isoquinoline alkaloids.     

Diversity of Babesia and Theileria species in symptomatic and asymptomatic dogs in Croatia

Babesiosis, the disease caused by tick-borne hematozoan parasites of the genus Babesia, is particularly common in dogs, and is caused by several “large” species of Babesia, as well as by an increasing number of “small” species of Babesia, some of which appear to be more closely related to members of the genus Theileria. In this work, blood samples were collected from 848 randomly selected, asymptomatic dogs and from 81 symptomatic dogs, microscopically positive for Babesia, and characterised by PCR and sequence analysis of a fragment of the ssrRNA gene. A prevalence of 3.42% (29 of 848) was found in asymptomatic dogs and sequence analysis revealed the presence of Babesia canis canis in 20 dogs (69%), Babesia gibsoni in six dogs (21%), Babesia canis vogeli in two dogs (7%) and Theileria annae in one dog (3%). In the group of symptomatic dogs, which were all positive by PCR, B. canis canis was the predominant species (78 dogs, or 96%), followed by single infections with B. canis vogeli, Babesia caballi and Theileria equi. Our study has confirmed that dogs are infected with a wide range of both large and small piroplasm species and subspecies, including B. caballi and T. equi, two parasites usually found in horses. The detection of the pathogenic species B. canis canis and B. gibsoni in asymptomatic dogs indicates that the relationship between parasite species/subspecies and clinical signs of infection in dogs deserves further investigation. Finally, the identities of the tick vectors transmitting T. annae and B. caballi remain to be elucidated.