Updating the East Asian mtDNA phylogeny: a prerequisite for the identification of pathogenic mutations

Knowledge about the world phylogeny of human mitochondrial DNA (mtDNA) is essential not only for evaluating the pathogenic role of specific mtDNA mutations but also for performing reliable association studies between mtDNA haplogroups and complex disorder

Antibacterial activity of a marine bacterium against pathogenic and environmental isolates of vibrio species

The marine environment covers three quarters of the surface of the planet is estimated to be home to more than 80% of life and yet it remains largely unexplored. The rich diversity of marine flora and fauna and its adaptation to the harsh marine environment coupled with new developments in biotechnology, has opened up a new exciting vista for extraction of bioactive products of use in medicine. In this study inhibitory activity of a marine bacterium isolated from gut of ribbonfish was studied against pathogenic and environmental isolates of Vibrio species. This strain was identified as Pseudomonas stutzeri and it was found active against V. harveyi (luminescent bacteria), V. cholerae, V. alginolyticus, V. damseal, V. fluvialis. The antibacterial substance produced by Pseudomonas stutzeri was soluble in organic solvent and closely bound to external surface of bacterial cells. Reduction of the absorbance of the V. cholera cell suspension was observed when log phase cells of V. cholerae were treated with MIC and 4xMIC concentration of crude extract of Pseudomonas stutzeri.

Phylogenetic positions of several amitochondriate protozoa - Evidence from phylogenetic analysis of DNA topoisomerase II

Several groups of parasitic protozoa, as represented by Giardia, Trichomonas, Entamoeba and Microsporida, were once widely considered to be the most primitive extant eukaryotic group - Archezoa. The main evidence for this is their 'lacking mitochondria' and possessing some other primitive features between prokaryotes and eukaryotes, and being basal to all eukaryotes with mitochondria in phylogenies inferred from many molecules. Some authors even proposed that these organisms diverged before the endosymbiotic origin of mitochondria within eukaryotes. This view was once considered to be very significant to the study of origin and evolution of eukaryotic cells (eukaryotes). However, in recent years this has been challenged by accumulating evidence from new studies. Here the sequences of DNA topoisomerase 11 in G lamblia, T vaginalis and E histolytica were identified first by PCR and sequencing, then combining with the sequence data of the microsporidia Encephalitozoon cunicul and other eukaryotic groups of different evolutionary positions from GenBank, phylogenetic trees were constructed by various methods to investigate the evolutionary positions of these amitochondriate protozoa. Our results showed that since the characteristics of DNA topoisomerase 11 make it avoid the defect of 'long-branch attraction' appearing in the previous phylogenetic analyses, our trees can not only reflect effectively the relationship of different major eukaryotic groups, which is widely accepted, but also reveal phylogenetic positions for these amitochondriate protozoa, which is different from the previous phylogenetic trees. They are not the earliest-branching eukaryotes, but diverged after some mitochondriate organisms such as kinetoplastids and mycetozoan; they are not a united group but occupy different phylogenetic positions. Combining with the recent cytological findings of mitochondria-like organelles in them, we think that though some of them (e.g. diplomonads, as represented by Giardia) may occupy a very low evolutionary position, generally these organisms are not as extremely primitive as was thought before; they should be polyphyletic groups diverging after the endosymbiotic origin of mitochondrion to adapt themselves to anaerobic parasitic life.

Relationships between Testate Amoebae Assemblages (Protozoa) and Geographic Factors in Yunnan Plateau Lakes, China

Knowledge of the effect of geographic factors on the assemblages of protozoan testate amoebae is still limited, despite there having been a number of studies on this fauna. We applied statistical analyses to data on the distribution of testate amoebae from nine major lakes in the Yunnan Plateau, southwest China. Cluster analysis, based on community structure, separated the lakes into two groups - the oligotrophic/mesotrophic lakes and the hypercutrophic lakes - confirming the idea that the testate amoebae assemblages in lakes are closely related to the trophic status. Additionally, within the oligotrophic/mesotrophic lakes, there was distinct geographic clustering. Linear regression analysis and the Mantel test both revealed that similarity of species composition decreased with increasing geographic distance among the oligotrophic/mesotrophic lakes.

Temporal and spatial variations in the species composition, distribution, and abundance of protozoa in East Dongting lake, China

Investigations of protozoa were carried out during four surveys of East Dongting Lake, China. A total of 160 protozoan species belonging to 71 genera was identified, of which 53 were flagellates, 37 sarcodines, and 70 ciliates. Among them, Peritrichida (32.6% of frequency), Arcellinida (16.2%), Volvocales (13.61/6), Peridiniales (13.1%), and Chrysomonadales (9.1%) were the main groups and contributed to 84.5% of the overall species. Ciliates were mainly composed of sessile species and small species. The total protozoan abundance varied from 2,400 cells L-1 to 20,250 cells L-1. The highest protozoan abundance occurred in spring; the lowest number was in autumn. The highest abundance of ciliates occurred in spring and winter, whereas flagellates developed the highest abundance in,summer and autumn. Pearson correlation analysis and linear regressions indicated that chlorophyll a and water velocity were the main factors affecting ternporal and spatial variations of the protozoan abundance.

The temporal and spatial distribution, composition and abundance of Protozoa in Chaohu Lake, China: Relationship with eutrophication

Systematic investigations into the temporal and spatial distribution, composition and abundance of protozoa in two regions with different trophic levels in Chaohu Lake, a large, shallow and highly eutrophic freshwater lake in China, were conducted during 2002-2003. A total of 114 species of protozoa, including phytomastigophorans, zoomastigophorans, amoebae and ciliates, were identified from 120 polyurethane foam unit (PFU) samples exposed at four stations and from various types of natural substrates. Of the 114 taxa, 36 core species were found on PFU substrates and 23 of these were found on natural ones. Protozoan abundance and chemical physical parameters at nine sampling stations, four in the western lake and five in the eastern part, indicate trophic gradient changes along the lake. Seasonal variations in the species composition of major groups at littoral PFU sampling stations illustrate the effect of a severe algal bloom on the protozoan community structure. Temporal and spatial distributions of individual abundance as functions of water temperature and trophic status were revealed. This study demonstrates again that the PFU artificial substrate method samples protozoan communities more effectively than routine natural substrate methods. (c) 2005 Elsevier GmbH. All rights reserved.

Use of the aquatic protozoa to formulate a community biotic index for an urban water system

Protozoan were collected from 16 stations in water system of Changde City (China) using the PFU method. Sampling programs were conduced on a yearly basis, with seasonal frequency at diverse sites in the water system and 488 species of protozoa was identified. At the same time, Water sampling from these stations was conducted and various water chemical parameters, including DO, COD, BOD5, NH3, TP, and Volatile Phenol, were analyzed. The aim of the research was, on one hand, using chemical method to take an investigation to the water pollution status of Changde City; on the other hand, using protozoan to make an evaluation to the water quality. With the chemical water parameters and protozoa data, a biotic index was derived for the investigated region. The species pollution value (SPV) of 469 protozoa species was established, and the community pollution value (CPV) calculated from SPV was used to evaluate water quality. The method of the biotic index was tested and the result showed that CPV calculated from SPV had a close correlation with the degree of water pollution (p < 0.00001). This indicated that the method of the biotic index is reliable. The water quality degrees divided by CPV were suggested. (c) 2004 Elsevier B.V. All rights reserved.

Testate amoebae (Protozoa : Rhizopoda) from northwest Yunnan, China

A survey of the testate amoebae of northwest Yunnan in the Hengduan Mountains region of southwest China revealed 151 species belonging to 29 genera. Of these, three genera and 27 species were new to the testate amoebae fauna of China. The widely distributed and dominant species were Centropyxis aculeata aculeata, C aerophila aerophila, C. sylvatica, Difflugia elegans, D. globulosa, D. pristis, D. pulex, Phryganella hemisphaerica, Trinema enchelys, and T lineare. The most diverse genera were Difflugia (41 species), Centropyxis (31 species), Arcella (18 species), and Euglypha (11 species). The number of species in each sample varied greatly. It appears that species diversity of testate amoebae is related not only to macro-environments (e.g., geographic zonation) but also to microenvironments (e.g., microhabitats).

Morphology, biometry and distribution of Difflugia biwae Kawamura, 1918 (Protozoa : Rhizopoda)

The freshwater testate amoeba Difflugia biwae Kawamura, 1918, isolated from Mulan Lake, Hubei Province, China is investigated using light microscopy and scanning electron microscopy. The morphology, biometry and distribution of this little known species are supplied. After careful comparison with three other similar species, including D. delicatula Gauthier-Livre et Thomas, D. elegans Penard and D. oblonga caudata Stepanek, we believe that the characteristics of smooth fusiform shell, conspicuous great collar flare (always larger than body-width) around the aperture, constriction behind the aperture and a somewhat curved aboral horn set D. biwae apart from all other Difflugia species. This species shows a great diversity in total length, collar height and aboral horn length which have high variability (CV between 11.76 and 24.52). However, body width, collar diameter, neck width, body length and aperture diameter are fairly constant with low variability (CV between 5.34 and 8.79) which shows a remarkable uniformity of D. biwae. Also, the size frequency distributions of both body width and body length yield bell-shaped (normally distributed) curves and indicate that D. biwae is a size-monomorphic species, characterized by a main-size class and a small size range. D. biwae is probably endemic to East Asia (China and Japan) because it has such a large size (165-306 mu m) that it would have been easily found in Europe and North America, if it were there. Consequently, D. biwae must have a restricted geographical distribution, disproving the old hypothesis that microscopic organisms are cosmopolitan.

Redescription of Difflugia tuberspinifera Hu, Shen, Gu et Gong, 1997 (Protozoa : Rhizopoda : Arcellinida : Difflugiidae) from China

The freshwater testate amoeba Difflugia tuberspinifera Hu et al. 1997 collected from pond and lake in China, is investigated by light and scanning electron microscopy. This little known taxon is redescribed and its morphology, biometry and ecology are supplied. After carefully comparison with other six similar species including Difflugia bartosi Stepanek, D. corona Wallich, D. corona cashi Deflandre, D. corona tuberculata Vucetich, D. muriformis Gauthier-Lievre et Thomas and Netzelia tuberculata (Wallich) Netzal we believe that the sub-spherical to spherical shell, the mulberry-shaped appearance, the 7-10 apertural tooth-like structures, the short collar and the conical spines numbering from 4 to 8 at the upper equatorial region in D. tuberspinifera set it apart from other species. Besides, statistical analysis indicates that D. tuberspinifera is a size-monomorphic species characterized by a main-size class and a small size range and the shell height is significant correlated with other morphometric characters at p < 0.05 excepting the number of aperture tooth-like structures and the number of spines. Moreover, D. tuberspinifera inhabits not only lotic but also lentic environment.

Infraciliature and myoneme system of Campanella umbellaria (Protozoa, Ciliophora, Peritrichida)

The infraciliature and myoneme system of Campanella umbellaria were revealed using the protargol impregnation technique. The main characteristics of the infraciliature are the peristomial ciliary rows (haplokinety and polykineties), which make four and a half turns around the peristomial disc before plunging into the infundibulum, and the aboral infraciliature, which is made up of the aboral ciliary wreath (trochal band) and the scopula. The myoneme system is composed of: 1) longitudinal fibers, which include 60-84 (mean 72.3) short longitudinal fibers, 40-56 (mean 45.8) medium-length longitudinal fibers, and numerous long longitudinal fibers; and 2) circular fibers, which include 8-12 (mean 9.3) peristomial ring fibers, linking fibers, support fibers, and peristomial disc fibers. The various fibers in C. umbellaria are interconnected to form a single myoneme system that may act as a cell skeleton as well as providing the mechanism by which the zooid contracts and relaxes. (C) 2004 Wiley-Liss, Inc.

Characterisation of a pathogenic virus isolated from marine threadfin fish (Eleutheronema tetradactylus) during a disease outbreak

An unknown virus was isolated from massive mortality of cultured threadfin (Eleutheronema tetradactylus) fingerlings. The virus replicated in BF-2 fish cell line and produced a plaque-like cytopathic effect. Electron micrographs revealed non-enveloped, icosahedral particles approximately 70-80 nm in diameter composed of a double capsid layer. Viroplasms and subviral particles approximately 30 run in diameter and complete particles of 70 nm in diameter were also observed in the infected BF-2 tissue culture cells. The virus was resistant upon pH 3 to 11 and ether treatment. It is also stable to heat treatment (3 h at 56 T). Replication was not inhibited by 5-iododeoxyuridine (5-IUdR). Acridine orange stain revealed typical reovirus-like cytoplasmic inclusion bodies. Electrophoresis of purified virus revealed 11 segments of double-stranded RNA and five major structural polypeptides of approximately 136, 132, 71, 41 and 33 kDa. Based on these findings, the virus isolated was identified to belong to the genus Aquareovirus and was designated as threadfin reovirus. This virus differed from a majority of other aquareovirus by its increase in virus infectivity upon exposure to various treatments such as high and low pH, heat (56 degreesC), ether and 5-IUdR. The RNA and virion protein banding pattern of the threadfin reovirus was shown to differ from another Asian isolate, the grass carp hemorrhage reovirus (GCV). Artificial injection of the threadfin reovirus into threadfin fingerlings resulted in complete mortality, whereas sea bass (Lates calcarifer) fingerlings infected via bath route showed severe mortality within a week after exposure. These results indicate that the threadfin virus is another pathogenic Asian aquareovirus isolate that could cross-infect into another marine fish, the sea bass. (C) 2002 Elsevier Science B.V. All rights reserved.

Analysis of the effect of copper on the virulence of a pathogenic Edwardsiella tarda strain

Aim: To investigate the effect of copper on the virulence of Edwardsiella tarda. Methods and Results: The pathogenic Edw. tarda strain TX5 was cultured under copper-stressed conditions and examined for any potential alteration in capacities that are associated with pathogenicity. The results showed that compared to untreated TX5, Cu-treated TX5 exhibits reduced planktonic and biofilm growth, an impaired ability to adhere to host mucus, modulation of host immune response, and dissemination in host blood and liver. Consistent with these observations, the overall bacterial virulence of Cu-treated TX5 is significantly attenuated. SDS-PAGE analyses of whole cell protein production showed that Cu-treated TX5 differs from the untreated TX5 in its production of at least one protein. Quantitative real time reverse transcriptase PCR analyses showed that copper treatment decreased the expression of virulence-associated genes encoding components of the type III and type VI secretion systems, the Eth haemolysin system, and the LuxS/AI-2 quorum-sensing system. Conclusions: Prolonged exposure to copper has multiple effects on TX5 and results in significant attenuation of bacterial virulence. Significance and Impact of the Study: The results of this study demonstrate that copper treatment has a broad and profound effect on the virulence-associated capacities of TX5, which is exerted at least in part at the transcription level. These findings provide new insights to the antimicrobial mechanism of copper.

Identification, Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain

A gene, pfa1, encoding an autotransporter was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased fish. The expression of pfa1 is enhanced during infection and is regulated by growth phase and growth conditions. Mutation of pfa1 significantly attenuates the overall bacterial virulence of TSS and impairs the abilities of TSS in biofilm production, interaction with host cells, modulation of host immune responses, and dissemination in host blood. The putative protein encoded by pfa1 is 1,242 amino acids in length and characterized by the presence of three functional domains that are typical for autotransporters. The passenger domain of PfaI contains a putative serine protease (Pap) that exhibits apparent proteolytic activity when expressed in and purified from Escherichia coli as a recombinant protein. Consistent with the important role played by PfaI in bacterial virulence, purified recombinant Pap has a profound cytotoxic effect on cultured fish cells. Enzymatic analysis showed that recombinant Pap is relatively heat stable and has an optimal temperature and pH of 50 degrees C and pH 8.0. The domains of PfaI that are essential to autotransporting activity were localized, and on the basis of this, a PfaI-based autodisplay system (named AT1) was engineered to facilitate the insertion and transport of heterologous proteins. When expressed in E. coli, AT1 was able to deliver an integrated Edwardsiella tarda immunogen (Et18) onto the surface of bacterial cells. Compared to purified recombinant Et18, Et18 displayed by E. coli via AT1 induced significantly enhanced immunoprotection.