Isolation of a vahlkampfiid amoeba from a contact lens: Tetramitus ovis (Schmidt, 1913) n. comb.

A vahlkampfiid amoeba has been isolated from a contact lens. Based on 5.8S rDNA sequence analysis the strain belongs to the genus Tetramitus. The internal transcribed spacer (ITS1 and ITS2) sequences differ by 3 and 9 bp, respectively, from T. lobospinosus. The morphology of the cyst does not correspond to T. lobospinosus, but is identical to that published for Vahlkampfia ovis at the beginning of the last century. There is no reference strain of V. ovis to investigate using molecular techniques. Therefore, we propose that the strain under study should be considered to represent the neotype strain of V. ovis, now classified as T. ovis (Schmidt, 1913) n. comb.     

Two new Tetramitus species (Heterolobosea, Vahlkampfiidae) from cold aquatic environments

Characterisation of the protists of cold environments provides important background for assessing the effects of climate change on microbial communities. Tetramitus angularis n. sp., from aquatic environments in Iceland and Switzerland, is the first vahlkampfiid recognised to have a characteristic Tetramitus flagellate stage combined with pre-formed excystment pores, which are not typical of this genus. T. angularis amoebae have a typical vahlkampfiid locomotive form and contain prominent lipid inclusions. Flagellates have a collar and cytostome, and can be mono- to multi-nucleate with corresponding change in cell shape from cylindrical to ellipsoidal and variable number of flagella. Cysts are round to semi-angular and have 2-5 pores closed by protruding, translucent plugs. A second organism, T. parangularis n. sp. from Alaska, has similar cysts but a flagellate stage has not been recognised; ITS sequence divergence is consistent with species criteria in the Vahlkampfiidae. Phylogenetic analysis of sequence data for the 5.8S rDNA region clusters the new spp. with T. rostratus, T. entericus and T. waccamawensis.     

Analysis of internal transcribed spacer1 (ITS1) region of rDNA for genetic characterization of Paramphistomum sp.

Paramphistomosis is the most prevalent disease of domestic ruminants, causing heavy economic loss in many countries across the world. The morphological identification of these parasites is difficult, therefore molecular characterization is used to discriminate Paramphistomum species. The present study was conducted to identify Paramphistomum sp. at Mardan District, Khyber Pakhtunkhwa (KPK), Pakistan. All samples of these rumen flukes were collected from buffalo. The gDNA was isolated from the adult parasites and the ITS1 region was amplified for the sequence analysis. All flukes had 100% similarity and there was no intraspecific variation. The Blast results showed that all flukes were P. cervi as they form a single cluster with P. cervi reported from China. The results of the ITS1 sequences of the present study with reference sequencing from China showed eight specific SNPs. This was the first study in which P. cervi was genetically characterized through the ITS1 region of rDNA at District Mardan, Pakistan. It can also be used as a marker for the genetic identification of Paramphistomum species.     

Morphology and phylogeny of Bryophryoides ocellatus n. g., n. sp. (Ciliophora,Colpodea) from in situ soil percolates of Idaho,U.S.A.

We describe the morphology and 18S rDNA phylogeny of Bryophryoides ocellatus n. g., n. sp., a bryophryid ciliate inhabiting in situ soil percolates from Idaho, U.S.A. The new genus is distinguished from other bryophryid genera by a combination of the following features: (1) kreyellid (irregularly meshed) silverline pattern, (2) polymorphic adoral organelles in the preoral suture, (3) absence of vestibular kineties. In phylogenetic analyses, Bryophryoides ocellatus is most closely related to Bryophrya gemmea. The 18S rDNA sequence pairwise distance of 2% between these genera, while similar to that between many colpodidan species, exceeds that between some colpodidan genera (e.g. Mykophagophrys and Pseudoplatyophrya, 1.1%), further supporting establishment of the new genus. Topology hypothesis testing strongly supports the monophyly of the Colpodida including the bryophryids. Despite weak nodal support, tests of topology constraints narrowly reject the non-monophyly of the sequenced Bryophryidae (Bryophrya + Bryophryoides + Notoxoma). Likewise, the monophyletic origin of the sequenced Bryophryidae is indicated in the phylogenetic networks though with low support.     

Determination of internal transcribed spacer regions (ITS) in Trichomonas vaginalis isolates and differentiation among Trichomonas species

The nucleotide sequence of the 5.8S rRNA gene and the flanked internal transcribed spacer (ITS) regions of six Trichomonas vaginalis isolates with different metronidazole sensitivity and geographic origin were genotyped. A multiple sequence alignment was performed with different sequences of other isolates available at the GenBank/EMBL/DDBJ databases, which revealed 5 different sequence patterns. Although a stable mutation in position 66 of the ITS1 (C66T) was observed in 26% (9/34) of the T. vaginalis sequences analyzed, there was 99.7% ITS nucleotide sequence identity among isolates for this sequence. The nucleotide sequence variation among other species of the genus Trichomonas ranged from 3.4% to 9.1%. Surprisingly, the % identity between T. vaginalis and Pentatrichomonas hominis was ~ 83%. There was > 40% divergence in the ITS sequence between T. vaginalis and Tritrichomonas spp., including Tritrichomonas augusta, Tritrichomonas muris, and Tritrichomonas nonconforma and with Tetratrichomonas prowazeki. Dendrograms grouped the trichomonadid sequences in robust clades according to their genera. The absence of nucleotide divergence in the hypervariable ITS regions between T. vaginalis isolates suggests the early divergence of the parasite. Importantly, these data show this ITS1-5.8S rRNA-ITS2 region suitable for inter-species differentiation.     

Secondary structural modeling of the second internal transcribed spacer (ITS2) from Pfiesteria-like dinoflagellates (Dinophyceae)

Pfiesteria piscicida is a harmful bloom-forming alga that has received a great deal of attention due to its potential association with large fish kills and neurological problems in humans. Since the discovery of Pfiesteria, several other Pfiesteria-like dinoflagellates (PLDs) have also been identified. Genetic identification and phylogenetic relationships among the PLDs commonly utilize sequence data from the genes and spacers of the ribosomal DNA (rDNA) operon. Of these, the internal transcribed spacers (ITSs) have been previously shown to fold into secondary structures that are critical for proper ribosomal processing. In this study, we modeled the secondary structure of the second internal transcribed spacer (ITS2) from 16 PLDs (as well as an outgroup taxon) using phylogenetic comparative methods and minimum free energy. The secondary structural models predicted for these dinoflagellates consisted of four paired helices separated by five unpaired regions, consistent with those reported from many eukaryotes. All of the structures were highly stable (ΔG = ?66.1 to ?122.3 kcal·mol at 37 °C) and several structural characters were found to be conserved either across the PLDs or were specific to monophyletic subgroups, strengthening previously inferred phylogenetic relationships among taxa. Additionally, an 18 bp motif was identified in the PLDs whose position corresponds to a ribosomal processing site described from other eukaryotes. Potential applications of these ITS2 secondary structures include utility in strain and species identification, phylogenetic inference and serving as a tool for identifying and excluding rDNA pseudogenes when assessing biodiversity within the PLDs.     

Isolation and identification of a novel Rhodococcus sp. ML-0004 producing epoxide hydrolase and optimization of enzyme production

Rhodococcus sp. ML-0004, a novel strain for producing epoxide hydrolase, was isolated from soil in this study. The epoxide hydrolase can catalyze the stereo-specific hydrolysis of cis-epoxysuccinic acid to generate l(+)-tartaric acid. By examining physiological, biochemical characteristics and comparing its 16S rDNA gene sequence, it was identified as Rhodococcus opacus, and named R. opacus ML-0004. The optimal conditions for epoxide hydrolase production from R. opacus ML-0004 were also investigated. Propanediol and (NH₄)₂SO₄ were selected as carbon source and nitrogen source, respectively, for the production of R. opacus ML-0004 epoxide hydrolase. The optimal conditions for epoxide hydrolase production were fermentation temperature = 28 °C, pH 7.0, and cultivation time = 26 h. Under these conditions, the maximum epoxide hydrolase activity reached 10.5 U mL⁻¹.     

New record of Ascaridia nymphii (Secernentea: Ascaridiidae) from macaw parrot,Ara chloroptera,in China

Present study was performed to identify the species of ascarids from macaw parrot, Ara chloroptera, in China. Total 6 ascarids (3 males and 3 females) were collected in the feces of 3 macaws at Guangzhou Zoo in Guangdong Province, China. Their morphological characteristics with dimensions were observed under a light microscope, and their genetic characters were analyzed with the partial 18S rDNA, ITS rDNA and nad4 gene sequences, respectively. Results showed that all worms have no interlabia but male worms have two alate spicules, well-developed precloacal sucker and a tail with ventrolateral caudal alae and 11 pairs of papillae. The partial 18S rDNA, ITS rDNA and nad4 sequences were 831 bp, 1015 bp and 394 bp in length, respectively. They showed the highest similarity of 99.8% (18S rDNA) with Ascaridia nymphii, 93.8% identities (ITS rDNA) with A. columbae and 98.5% to 99.5% identities (nad4) with Ascaridia sp. from infected parrot. All Ascaridia nematodes from the macaws were clustered into one clade and formed monophyletic group of Ascaridia with A. columbae and A. galli in two phylogenetic trees. It is observed that the combining morphological and sequencing data from three loci, the present Ascaridia species was identified as Ascaridia nymphii, which is the first record of A. nymphii from macaw parrot in China.     

Morphological and molecular characterization of Thelohanellus macrovacuolaris n. sp. (Myxosporea: Bivalvulida) infecting the palate in the mouth of common carp Cyprinus carpio L. in China

Thelohanellus macrovacuolaris n. sp. is described during a survey on myxozoan diversity of common carp Cyprinus carpio L. in China. It is characterized by the presence of round or ellipsoidal plasmodium in the palate in the mouth of host. Mature spores were pyriform in frontal view, lemon shaped in lateral view, measuring 21.6 ± 0.9 (19.3–23.8) long, 12.5 ± 0.7 (10.3–13.6) wide, and 10.2 ± 0.4 (9.8–11.8) thick. Most spores were surrounded by the membrane sheath. Single polar capsule was round with an apophysis at its top end presented close to apex of spore, measuring 9.1 ± 0.6 (8.0–10.0) in length, 8.6 ± 0.5 (7.8–9.6) in width. Polar filaments coiled, with 7 to 8 turns. A large, round iodinophilous vacuole was present, with 5.8–7.5 in diameter. The present species is morphologically distinct from all other Thelohanellus species. The BLAST search indicated that the newly obtained small subunit ribosomal RNA (ssrRNA) gene sequence of T. macrovacuolaris n. sp. did not match any available sequences in GenBank. Phylogenetically, T. macrovacuolaris n. sp. was placed sister to Thelohanellus wangi in the Thelohanellus clade. Both morphology and ssrRNA gene sequence data revealed that the present parasite is a new species of genus Thelohanellus.     

Bacterial decolorization and degradation of the reactive dye Reactive Red 180 by Citrobacter sp. CK3

A bacterial strain, CK3, with remarkable ability to decolorize the reactive textile dye Reactive Red 180, was isolated from the activated sludge collected from a textile mill. Phenotypic characterization and phylogenetic analysis of the 16S rDNA sequence indicated that the bacterial strain belonged to the genus Citrobacter. Bacterial isolate CK3 showed a strong ability to decolorize various reactive textile dyes, including both azo and anthraquinone dyes. Anaerobic conditions with 4 g l^?1 glucose, pH = 7.0 and 32 °C were considered to be the optimum decolorizing conditions. Citrobacter sp. CK3 grew well in a high concentration of dye (200 mg l^?1), resulting in approximately 95% decolorization extent in 36 h, and could tolerate up to 1000 mg l^?1 of dye. UV–vis analyses and colorless bacterial cells suggested that Citrobacter sp. CK3 exhibited decolorizing activity through biodegradation, rather than inactive surface adsorption. It is the first time that a bacterial strain of Citrobacter sp. has been reported with decolorizing ability against both azo and anthraquinone dyes. High decolorization extent and facile conditions show the potential for this bacterial strain to be used in the biological treatment of dyeing mill effluents.     

Fibrophrys columna gen. nov., sp. nov: A member of the family Amphifilidae

A novel Diplophrys-like organism, Fibrophrys columna, was isolated from Hiuchigaike Pond in Japan. F. columna showed a nearly orbicular or broadly elliptical cell shape and has fine filamentous, branching ectoplasmic elements emanating from both polar ends of the cell. Cells also contain orange, amber, or colorless lipid bodies. Although its whole cell morphology resembles that of the genus Diplophrys, Fibrophrys is clearly distinct from Diplophrys on the basis of 18S rDNA sequences. Molecular phylogenetic analysis showed a close relationship of F. columna with Amphifila marina, and its sequence is similar to many environmental stramenopile sequences. The cells of F. columna measured 5.0–8.3 × 5.6–10.3 μm and sometimes possessed hernia-like prongs instead of filamentous ectoplasmic elements. An axis-like electron-dense body was observed in the mitochondria. We also studied the ultrastructure of another Fibrophrys strain, Fibrophrys sp. E-1, which is different from the type strain of F. columna. A ladder-like pattern was recognized in the outer part of unidentified cytoplasmic membranes connected with the mitochondria. The unidentified cytoplasmic membranes were connected to the nuclear, lipid body, and mitochondrial outer membranes. We propose a new genus, Fibrophrys, and a new species, F. columna, based on these ultrastructural and molecular features.     

Rahnella victoriana sp. nov., Rahnella bruchi sp. nov., Rahnella woolbedingensis sp. nov., classification of Rahnella genomospecies 2 and 3 as Rahnella variigena sp. nov. and Rahnella inusitata sp. nov., respectively and emended description of the genus Rahnella

Isolations from oak symptomatic of Acute Oak Decline, alder and walnut log tissue, and buprestid beetles in 2009–2012 yielded 32 Gram-negative bacterial strains showing highest gyrB sequence similarity to Rahnella aquatilis and Ewingella americana. Multilocus sequence analysis (using partial gyrB, rpoB, infB and atpD gene sequences) delineated the strains into six MLSA groups. Two MLSA groups contained reference strains of Rahnella genomospecies 2 and 3, three groups clustered within the Rahnella clade with no known type or reference strains and the last group contained the type strain of E. americana. DNA–DNA relatedness assays using both the microplate and fluorometric methods, confirmed that each of the five Rahnella MLSA groups formed separate taxa. Rahnella genomospecies 2 and 3 were previously not formally described due to a lack of distinguishing phenotypic characteristics. In the present study, all five Rahnella MLSA groups were phenotypically differentiated from each other and from R. aquatilis. Therefore we propose to classify the strains from symptomatic oak, alder and walnut and buprestid beetles as: Rahnella victoriana sp. nov. (type strain FRB 225^T = LMG 27717^T = DSM 27397^T), Rahnella variigena sp. nov. (previously Rahnella genomosp. 2, type strain CIP 105588^T = LMG 27711^T), Rahnella inusitata sp. nov. (previously Rahnella genomosp. 3, type strain DSM 30078^T = LMG 2640^T), Rahnella bruchi sp. nov. (type strain FRB 226^T = LMG 27718^T = DSM 27398^T) and Rahnella woolbedingensis sp. nov. (type strain FRB 227^T = LMG 27719^T = DSM 27399^T).     

Agrobacterium bohemicum sp. nov. isolated from poppy seed wastes in central Bohemia

Two non-pathogenic strains R89-1 and R90^T isolated from poppy seed (Papaver somniferum L.) wastes were phenotypically and genotypically characterized. Multilocus sequence analysis (MLSA) was conducted with six genes (atpD, glnA, gyrB, recA, rpoB, 16S rRNA). The strains represented a new species which clustered with Agrobacterium rubi NBRC 13261^T and Agrobacterium skierniewicense Ch11^T type strains. MLSA was further accompanied by whole-genome phylogeny, in silico DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses for both strains. ANI and dDDH values were deep below the species delineation threshold. Phenotypic features of the novel strains unequivocally allowed their differentiation from all other Agrobacterium species. Unlike other agrobacteria, the strains were salt sensitive and were able to biotransform morphine alkaloids. The dominant cellular fatty acids are 18:1 w7c, 16:0 and 12:0 aldehyde/16:1 iso I/14:0 3OH summed in feature 2 and the major respiratory quinine is Q-10 (87%). The DNA G + C content is 56 mol%. Microbial community analysis indicated probable association with P. somniferum plant material. Altogether, these characteristics showed that strains R90^T and R89-1 represent a new species of the genus Agrobacterium which we propose to name Agrobacterium bohemicum. The type strain of A. bohemicum is R90^T (=CCM 8736^T = DSM 104667^T).     

Mesorhizobium delmotii and Mesorhizobium prunaredense are two new species containing rhizobial strains within the symbiovar anthyllidis

Eight mesorhizobial symbiotic strains isolated from Anthyllis vulneraria root-nodules were studied and compared taxonomically with defined Mesorhizobium species. All strains presented identical 16S rDNA sequences but can be differentiated by multilocus sequence analysis of housekeeping genes (recA, atpD, glnII and dnaK). Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analyses separate these strains in two groups and a separate strain. Levels of DNA–DNA relatedness were less than 55% between representative strains and their closest Mesorhizobium reference relatives. The two groups containing four and three strains, respectively, originating from border mine and non-mining areas in Cévennes, were further phenotypically characterized. Groupings were further supported by average nucleotide identity values based on genome sequencing, which ranged from 80 to 92% with their close relatives and with each other, confirming these groups represent new Mesorhizobium species. Therefore, two novel species Mesorhizobium delmotii sp. nov. (type strain STM4623^T = LMG 29640^T = CFBP 8436^T) and Mesorhizobium prunaredense sp. nov. (type strain STM4891^T = LMG 29641^T = CFBP 8437^T) are proposed. Type strains of the two proposed species share accessory common nodulation genes within the new symbiovar anthyllidis as found in the Mesorhizobium metallidurans type strain.     

Rhizobium cauense sp. nov., isolated from root nodules of the herbaceous legume Kummerowia stipulacea grown in campus lawn soil

Three bacterial isolates (CCBAU 101002^T, CCBAU 101000 and CCBAU 101001) originating from root nodules of the herbaceous legume Kummerowia stipulacea grown in the campus lawn of China Agricultural University were characterized with a polyphasic taxonomic approach. Comparative 16S rRNA gene sequence analysis showed that the isolates shared 99.85–99.92% sequence similarities and had the highest similarities to the type strains of Rhizobium mesoamericanum (99.31%), R. endophyticum (98.54%), R. tibeticum (98.38%) and R. grahamii (98.23%). Sequence similarity of four concatenated housekeeping genes (atpD, glnII, recA and rpoB) between CCBAU 101002^T and its closest neighbor (R. grahamii) was 92.05%. DNA–DNA hybridization values between strain CCBAU 101002^T and the four type strains of the most closely related Rhizobium species were less than 28.4 ± 0.8%. The G + C mol% of the genomic DNA for strain CCBAU 101002^T was 58.5% (Tm). The major respiratory quinone was ubiquinone (Q-10). Summed feature 8 (18:1ω7cis/18:1ω6cis) and 16:0 were the predominant fatty acids. Strain CCBAU 101002^T contained phosphatidylcholine and phosphatidylethanolamine as major polar lipids, and phosphatidylglycerol and cardiolipin as minor ones. No glycolipid was detected. Unlike other strains, this novel species could utilize dulcite or sodium pyruvate as sole carbon sources and it was resistant to 2% (w/v) NaCl. On the basis of the polyphasic study, a new species Rhizobium cauense sp. nov. is proposed, with CCBAU 101002^T (=LMG 26832^T = HAMBI 3288^T) as the type strain.     

A New Heterolobosean Amoeboflagellate,Tetramitus dokdoensis n. sp., Isolated from a Freshwater Pond on Dokdo Island in the East Sea,Korea

The genus Tetramitus is a representative amoeboflagellate group within the Heterolobosea, and currently contains over a dozen species. Here, a new heterolobosean amoeboflagellate was isolated from a freshwater pond on Dokdo Island, Korea. The amoebae have eruptive pseudopodia, no uroidal filament, and a nucleus with a central nucleolus. The length and width of the amoebae are 15.5–28.0 μm and 5.4–12.6 μm, respectively. The flagellates are conical, with 4 flagella of equal length (~10 μm). There is a discrete rostrum in the subapical region of the flagellate form. The cyst has thin endo‐ and ectocyst layers and no cyst pores. The amoeba shows slow movement at 37 °C, but does not move at 42 °C under a light microscope. Phylogenies of the 18S rRNA gene and the ITS1‐5.8S rRNA gene‐ITS2 sequence show that the strain belongs to a subclade of Tetramitus that includes Tetramitus rostratus, Tetramitus waccamawensis and Tetramitus entericus, amongst others. Nonetheless, the strain is distinct from other species in both molecular phylogenetic trees. Thus the strain isolated from the Dokdo Island is proposed as a novel species, Tetramitus dokdoensis n. sp.     

Morphological,ultrastructural and phylogenetic analyses of Myxobolus hilarii n. sp. (Myxozoa,Myxosporea), a renal parasite of farmed Brycon hilarii in Brazil

Myxobolus hilarii n. sp. was described, based on morphology, histology, ultrastructure and 18S rDNA sequencing, infecting the kidney of Brycon hilarii (Valenciennes 1850) (Characiformes: Bryconidae) taken from fish farms in the state of São Paulo, Brazil. Thirteen specimens of B. hilarii were examined and 100% had round, white plasmodia in the kidney. The mature myxospores were rounded, measuring 11.5 ± 0.8 (9.8–13.4) μm in length, 11.0 ± 0.7 (9.7–12.4) μm in width and 7.6 ± 1.0 (6.7–9.0) μm in thickness. Polar capsules were elongated and of equal size, with 6.5 ± 0.4 (6.0–7.2) μm in length and 4.0 ± 0.2 (3.6–5.3) μm in width and their polar filaments had 5 to 7 coils. Histological analysis revealed plasmodial development in the renal tubules, causing compression and deformation of adjacent tissues and destruction of renal tubule cells. Ultrastructural analysis showed direct contact between the plasmodial wall and the host tissue and asynchronous plasmodial development. The phylogenetic analysis of South American myxobolids, based on 18S rDNA sequencing, showed the myxosporeans grouping into two main clades. M. hilarii n. sp. appears as sister species of Myxobolus piraputangae.     

Purification and characterization of a thermostable endo-β-1,4-glucanase from a novel strain of Penicillium purpurogenum

A novel endo-β-1,4-glucanase (EG)-producing strain was isolated and identified as Penicillium purpurogenum KJS506 based on its morphology and internal transcribed spacer (ITS) rDNA gene sequence. P. purpurogenum produced one of the highest levels of EG (5.6 U mg-protein^?1) with rice straw and corn steep powder as carbon and nitrogen sources, respectively. The extracellular EG was purified to homogeneity by sequential chromatography of P. purpurogenum culture supernatants on a DEAE sepharose column, a gel filtration column, and then on a Mono Q column with fast protein liquid chromatography. The purified EG was a monomeric protein with a molecular weight of 37 kDa and showed broad substrate specificity with maximum activity towards lichenan. P. purpurogenum EG showed t_1/2 value of 2 h at 70 °C and catalytic efficiency of 118 ml mg^?1 s^?1, one of the highest levels seen for EG-producing microorganisms. Although EGs have been reported elsewhere, the high catalytic efficiency and thermostability distinguish P. purpurogenum EG.     

Intraspecific differentiation of Paramecium novaurelia strains (Ciliophora,Protozoa) inferred from phylogenetic analysis of ribosomal and mitochondrial DNA variation

Paramecium novaurelia Beale and Schneller, 1954, was first found in Scotland and is known to occur mainly in Europe, where it is the most common species of the P. aurelia complex. In recent years, two non-European localities have been described: Turkey and the United States of America. This article presents the analysis of intraspecific variability among 25 strains of P. novaurelia with the application of ribosomal and mitochondrial loci (ITS1-5.8S-ITS2, 5′ large subunit rDNA (5′LSU rDNA) and cytochrome c oxidase subunit 1 (COI) mtDNA). The mean distance observed for all of the studied P. novaurelia sequence pairs was p = 0.008/0.016/0.092 (ITS1-5.8S-ITS2/5′LSU rDNA/COI). Phylogenetic trees (NJ/MP/BI) based on a comparison of all of the analysed sequences show that the studied strains of P. novaurelia form a distinct clade, separate from the P. caudatum outgroup, and are divided into two clusters (A and B) and two branches (C and D). The occurrence of substantial genetic differentiation within P. novaurelia, confirmed by the analysed DNA fragments, indicates a rapid evolution of particular species within the Paramecium genus.