Development of a rapid, sensitive and specific diagnostic assay for fish Aquareovirus based on RT-PCR

A rapid, sensitive and highly specific detection method for Aquareovirus based on reverse-transcription polymerase chain reaction (RT-PCR) was developed. Based on multiple sequence alignment of the cloned sequences of a local isolates, the Threadfin reovirus (TFV) and Guppy reovirus (GPV) with Grass carp reovirus (GCRV), a pair of degenerate primers was selected carefully and synthesized. Using this primer combination, only one specific product, approximately 450 bp in length was obtained when RT-PCR was carried out using the genomic double-stranded RNA (dsRNA) of TFV, GPV and GCRV. Similar results were also obtained when Chum salmon reovirus (CSRV) and Striped bass reovirus (SBRV) dsRNA were used as templates. No products were observed when nucleic acids other than the dsRNA of the aquareoviruses described above were used as RT-PCR templates. This technique could detect not only TFV but also GPV and GCRV in low titer virus-infected cell cultured cells. Furthermore, this method has also been shown to be able to diagnose GPV-infected guppy (Poecilia reticulata) that exhibit clinical symptoms as well as GPV-carrier guppy. Collectively, these results showed that the RT-PCR amplification method using specific degenerate primers described below is very useful for rapid and accurate detection of a variety of aquareovirus strains isolated from different host species and origin. (C) 2004 Elsevier B.V. All rights reserved.

Optical Diagnostic and Modeling Solution Growth Process of Sodium Chlorate Crystals

Both a real time optical interferometric experiment and a numerical simulation of two-dimension non-steady state model were employed to study the growth process of aqueous sodium chlorate crystals. The parameters such as solution concentration distribution, crystal dimensions, growth rate and velocity field were obtained by both experiment and numerical simulation. The influence of earth gravity during crystal growth process was analyzed. A reasonable theory model corresponding to the present experiment is advanced. The thickness of concentration boundary layer was investigated especially. The results from the experiment and numerical simulation match well.

A diagnostic system for the microgravity experiments on Marangoni drop migrations

An optical diagnostic system designed for the microgravity experiments on Marangoni drop migrations has been depicted in the presented paper. One part of the optical system was used to image and record the drops tracks; the other part was an equal-thick interferential system, it has the ability to observe the fine structures of the drop migrations. Some ground-based experiments had been performed and the results were simply discussed in the present paper.

Isolation and identification of a novel WSSV nucleocapsid protein by cDNA phage display using an scFv antibody

In a previous study, a scFv phage display library against white spot syndrome virus (WSSV) was constructed and yielded a clone designated A I with conformational specificity against native but not denatured viral antigen. Although the clone A1 has been used successfully as a diagnostic antibody, its precise target antigen has not been elucidated. A different strategy was adopted involving the construction of a second T7 phage display library utilizing mRNA isolated from shrimp infected with WSSV. Following RT-PCR and T7 phage library construction, phages displaying the candidate epitope were selected with A I scFv. Since successive enrichment steps were not associated with an increased titer of the phages, enrichment after successive tests was confirmed by PCR resulting in the prefer-red selection of a specific DNA sequence encoding a novel nucleocapsid protein WSSV388. Immune electron microscopy revealed that WSSV388 is located on the nucleocapsid. This result demonstrated that unknown antigen could be identified by phage display using the epitope conformation dependent scFv. (c) 2006 Elsevier B.V. All rights reserved.

Phylogenetic relationships among Trichodinidae (Ciliophora : Peritricha) derived from the characteristic values of denticles

The phylogenetic relationships among trichodinids remain obscure. As an important diagnostic marker, the morphology of the denticles in the adhesive disc as well as the adoral spiral has been widely used in generic discrimination and species identification of trichodinids. We studied the characters of denticles of the ten genera of Trichodinidae and the sole genus Urceolaria of Urceolariidae by using a quantitative method. The characteristic values were used to generate Manhattan distance, on which the dendrogram was based to construct with the Unweighted Paired Group Method using the Arithmetic mean (UPGMA). The investigations show that all the genera of the family Trichodinidae were clearly separate from the outgroup Urceolaria, and within the Trichodinidae: (i) Dipartiella grouped with Trichodinella and Tripartiella and lay in the closest position to the outgroup with a low dissimilarity, suggesting Dipartiella might be the most primitive genus in the family; (ii) Hemitrichodina clustered in a single clad and lay in the farthest position to the outgroup with the highest dissimilarity, indicating that it might be the most advanced genus; and (iii) the other 6 genera, Trichodina, Paratrichodina, Semitrichodina, Vauchomia, Pallitrichodina and Trichodoxa clustered in a big clad with very low dissimilarity, showing that they are closely related to each other. We discuss the evolutionary trend of the denticle and conclude that the denticles of the adhesive disc should be an apomorphic feature of the trichodinids and their changes could reflect the evolutionary tendencies of these ciliates.

Evaluation of Clonorchis sinensis recombinant 7-kilodalton antigen for serodiagnosis of clonorchiasis

The diagnostic applicability of the Clonorchis sinensis recombinant 7-kDa protein was evaluated. In enzyme-linked immunosorbent assays and immunoblots, the protein showed high sensitivities (81.3 and 71.9%, respectively) and specificities (92.6 and 89.7%, respectively) for sera obtained from various helminthic infections. Some paragonimiasis sera showed cross-reactions. The antigen might be valuable in the serodiagnosis of human clonorchiasis.

Description of two new myxosporean species parasitic in freshwater fishes from the Yangtze River in China

Two new species of myxosporeans (Myxosporea: Myxidiidae), Myxidium tuanfengensis sp. n. and Zschokkella saurogobionis sp. n., Parasitic in freshwater fishes collected from the Yangtze River of China are described in this paper. M. tuanfengensis was found in the liver parenchyma and intestine lumen of Leptobotia taeniops Sauvage, 1878, while Z. saurogobionis was found in the gall bladder of Saurogobio dumerili Bleeker, 1871. The diagnostic characters of M. tuanfengensis are: round or elliptical polysporous plasmodia averaging 118 mum in size; spore oval in frontal view with smooth surface and nearly spindle-shape in sutural view with slightly sinuous sutural ridge, averaging 19.5 x 9.75 x 8.9 mum in size; two large spherical polar capsules 6.8 mum in diameter, with polar filament wound in 4 to 5 coils. The diagnostic characters of Z. saurogobionis are: spore elliptical in both frontal and sutural view measuring 18.3 x 9.8 x 10.8 mum in size; fine sutural ridge in S-form, spore shell marked with 10 to 12 distinct lines paralleled with the sutural line; two spherical polar capsules, 6.7 mum in diameter, with polar filament in 5 coils.

Rhodococcus qingshengii sp nov., a carbendazim-degrading bacterium

A Gram-positive, aerobic, non-motile, mesophilic strain, djl-6(T), able to degrade carbendazim, was isolated from a carbendazim-contaminated soil sample from Jiangsu province, China. The taxonomic position of this isolate was analysed by using a polyphasic approach. Chemotaxonomic analysis including peptidoglycan type, diagnostic sugar composition, fatty acid profile, menaquinones, polar lipids and mycolic acids showed that the characteristics of strain djl-6(T) were in good agreement with those of the genus Rhodococcus. DNA-DNA hybridization showed that it had low genomic relatedness with Rhodococcus baikonurensis DSM 44587(T) (31.8%), Rhodococcus erythropolis DSM 43066(T) (23.8%) and Rhodococcus globerulus DSM 43954(T) (117.7%), the three type strains to which strain djl-6(T) was most closely related based on 16S rRNA gene sequence analysis (99.78, 99.25 and 98.91% similarity, respectively). Based on the phenotypic properties and DNA-DNA hybridization data, strain djl-6(T) (=CGMCC 1.6580(T) =KCTC 19205(T)) is proposed as the type strain of a novel Rhodococcus species, Rhodococcus qingshengii sp. nov.

Stable Isotope Probing:Linking Functional Activity to Specific Members of Microbial Communities

Linking organisms or groups of organisms to specific functions within natural environments is a fundamental challenge in microbial ecology. Advances in technology for manipulating and analyzing nucleic acids have made it possible to characterize the members of microbial communities without the intervention of laboratory culturing. Results from such studies have shown that the vast majority of soil organisms have never been cultured, highlighting the risks of culture-based approaches in community analysis. The development of culture-independent techniques for following the flow of substrates through microbial communities therefore represents an important advance. These techniques, collectively known as stable isotope probing (SIP), involve introducing a stable isotope-labeled substrate into a microbial community and following the fate of the substrate by extracting diagnostic molecular species such as fatty acids and nucleic acids from the community and determining which specific molecules have incorporated the isotope. The molecules in which the isotope label appears provide identifying information about the organism that incorporated the substrate. Stable isotope probing allows direct observations of substrate assimilation in minimally disturbed communities, and thus represents an exciting new tool for linking microbial identity and function. The use of lipids or nucleic acids as the diagnostic molecule brings different strengths and weaknesses to the experimental approach, and necessitates the use of significantly different instrumentation and analytical techniques. This short review provides an overview of the lipid and nucleic acid approaches, discusses their strengths and weaknesses, gives examples of applications in various settings, and looks at prospects for the future of SIP technology.

Diagnostic experiments for transport mechanisms of suspended sediment discharged from the Yellow River in the Bohai Sea

Five diagnostic experiments with a 3D baroclinic hydrodynamic and sediment transport model ECOMSED in couple with the third generation wave model SWAN and the Grant-Madsen bottom boundary layer model driven by the monthly sediment load of the Yellow River, were conducted to separately diagnose effects of different hydrodynamic factors on transport of suspended sediment discharged from the Yellow River in the Bohai Sea. Both transport and spatio-temporal distribution of suspended sediment concentration in the Bohai Sea were numerially simulated. It could be concluded that suspended sediment discharged from the Yellow River cannot be delivered in long distance under the condition of tidal current. Almost all of sediments from the Yellow River are deposited outside the delta under the condition of wind-driven current, and only very small of them are transported faraway. On the basis of wind forcing, sediments from the Yellow River are mainly transported north-northwestward, and others which are first delivered to the Laizhou Bay are continuously moved northward. An obvious 3D structure characteristic of sediment transport is produced in the wind-driven and tide-induced residual circulation condition. Transport patterns at all layers are generally consistent with circulation structure, but there is apparent deviation between the depth-averaged sediment flux and the circulation structure. The phase of temporal variation of sediment concentration is consistent with that of the bottom shear stress, both of which are proved to have a ten-day cycle in wave and current condition.

Screening of genes expressed in vivo after infection by Vibrio anguillarum M3

Aims: Genes uniquely expressed in vivo may contribute to the overall pathogenicity of an organism and are likely to serve as potential targets for the development of new vaccine. This study aims to screen the genes expressed in vivo after Vibrio anguillarum infection by in vivo-induced antigen technology (IVIAT). Methods and Results: The convalescent-phase sera were obtained from turbot (Scophthalmus maximus) survived after infection by the virulent V. anguillarum M3. The pooled sera were thoroughly adsorbed with M3 cells and Escherichia coli BL21 (DE3) cells. A genomic expression library of M3 was constructed and screened for the identification of immunogenic proteins by colony immunoblot analysis with the adsorbed sera. After three rounds of screening, 19 putative in vivo-induced (ivi) genes were obtained. These ivi genes were catalogued into four functional groups: regulator/signalling, metabolism, biological process and hypothetical proteins. Three ivi genes were insertion-mutated, and the growth and 50% lethal dose (LD50) of these mutants were evaluated. Conclusions: The identification of ivi genes in V. anguillarum M3 sheds light on understanding the bacterial pathogenesis and provides novel targets for the development of new vaccines and diagnostic reagents. Significance and Impact of the Study: To the best of our knowledge, this is the first report describing in vivo-expressed genes of V. anguillarum using IVIAT. The screened ivi genes in this study could be new virulent factors and targets for the development of vaccine, which may have implications for the development of diagnostic regents.

Screening of genes expressed in vivo after infection by Vibrio anguillarum M3

Aims: Genes uniquely expressed in vivo may contribute to the overall pathogenicity of an organism and are likely to serve as potential targets for the development of new vaccine. This study aims to screen the genes expressed in vivo after Vibrio anguillarum infection by in vivo-induced antigen technology (IVIAT). Methods and Results: The convalescent-phase sera were obtained from turbot (Scophthalmus maximus) survived after infection by the virulent V. anguillarum M3. The pooled sera were thoroughly adsorbed with M3 cells and Escherichia coli BL21 (DE3) cells. A genomic expression library of M3 was constructed and screened for the identification of immunogenic proteins by colony immunoblot analysis with the adsorbed sera. After three rounds of screening, 19 putative in vivo-induced (ivi) genes were obtained. These ivi genes were catalogued into four functional groups: regulator/signalling, metabolism, biological process and hypothetical proteins. Three ivi genes were insertion-mutated, and the growth and 50% lethal dose (LD50) of these mutants were evaluated. Conclusions: The identification of ivi genes in V. anguillarum M3 sheds light on understanding the bacterial pathogenesis and provides novel targets for the development of new vaccines and diagnostic reagents. Significance and Impact of the Study: To the best of our knowledge, this is the first report describing in vivo-expressed genes of V. anguillarum using IVIAT. The screened ivi genes in this study could be new virulent factors and targets for the development of vaccine, which may have implications for the development of diagnostic regents.