Modified Simplified Rate Equation Model of Flowing Chemical Oxygen-Iodine Laser and its Application

A modified simplified rate equation (RE) model of flowing chemical oxygen-iodine laser (COIL), which is adapted to both the condition of homogeneous broadening and inhomogeneous broadening being of importance and the condition of inhomogeneous broadening being predominant, is presented for performance analyses of a COIL. By using the Voigt profile function and the gain-equal-loss approximation, a gain expression has been deduced from the rate equations of upper and lower level laser species. This gain expression is adapted to the conditions of very low gas pressure up to quite high pressure and can deal with the condition of lasing frequency being not equal to the central one of spectral profile. The expressions of output power and extraction efficiency in a flowing COIL can be obtained by solving the coupling equations of the deduced gain expression and the energy equation which expresses the complete transformation of the energy stored in singlet delta state oxygen into laser energy. By using these expressions, the RotoCOIL experiment is simulated, and obtained results agree well with experiment data. Effects of various adjustable parameters on the performances of COIL are also presented.

Modified Simplified Rate Equation Model of Flowing Chemical Oxygen-Iodine Laser and its Application

A modified simplified rate equation (RE) model of flowing chemical oxygen-iodine laser (COIL), which is adapted to both the condition of homogeneous broadening and inhomogeneous broadening being of importance and the condition of inhomogeneous broadening being predominant, is presented for performance analyses of a COIL. By using the Voigt profile function and the gain-equal-loss approximation, a gain expression has been deduced from the rate equations of upper and lower level laser species. This gain expression is adapted to the conditions of very low gas pressure up to quite high pressure and can deal with the condition of lasing frequency being not equal to the central one of spectral profile. The expressions of output power and extraction efficiency in a flowing COIL can be obtained by solving the coupling equations of the deduced gain expression and the energy equation which expresses the complete transformation of the energy stored in singlet delta state oxygen into laser energy. By using these expressions, the RotoCOIL experiment is simulated, and obtained results agree well with experiment data. Effects of various adjustable parameters on the performances of COIL are also presented.

Scale-similarity model for Lagrangian velocity correlations in isotropic and stationary turbulence

A scale-similarity model for Lagrangian two-point, two-time velocity correlations LVCs in isotropic turbulence is developed from the Kolmogorov similarity hypothesis. It is a second approximation to the isocontours of LVCs, while the Smith-Hay model is only a first approximation. This model expresses the LVC by its space correlation and a dispersion velocity. We derive the analytical expression for the dispersion velocity from the Navier-Stokes equations using the quasinormality assumption. The dispersion velocity is dependent on enstrophy spectra and shown to be smaller than the sweeping velocity for the Eulerian velocity correlation. Therefore, the Lagrangian decorrelation process is slower than the Eulerian decorrelation process. The data from direct numerical simulation of isotropic turbulence support the scale-similarity model: the LVCs for different space separations collapse into a universal form when plotted against the separation axis defined by the model.

Identification and characterization of a MBP isoform specific to hypothalamus in orange-spotted grouper (Epinephelus coioides)

Myelin basic protein (MBP), as a major component of the myelin sheath, has been revealed to play an important role informing and maintaining myelin structure in vertebrate nervous system. In teleost, hypothalamus is an instinctive brain center and plays significant roles in many physiological functions, such as energy metabolism, growth, reproduction, and stress response. In comparison with other MBP identified in vertebrates, a smallest MBP is cloned and identified from the orange-spotted grouper hypothalamic cDNA plasmid library in this study. RT-PCR analysis and Western blot detection indicate that the EcMBP is specific to hypothalamus, and expresses mainly in the tuberal hypothalamus in adult grouper. Immunofluorescence localization suggests that EcMBP should be expressed by oligodendrocytes, and the expressing cells should be concentrated in hypothalamus and the area surrounding hypothalamus, such as NPOpc, VC, DP, NLTm, and NDLI The studies on EcMBP expression pattern and developmental behaviour in the brains of grouper embryos and larvae reveal that the EcMBP-expressing cells are only limited in a defined set of cells on the border of hypothalamus, and suggest that the EcMBP-expressing cells might be a subpopulation of oliaodendrocyte progenitor cells. This study not only identifies a smallest MBP isoform specific to hypothalamus that can be used as a molecular marker of oligodendrocytes in fish, but also provides new insights for MBP evolution and cellular distribution. (C) 2007 Elsevier B.V.. All rights reserved.

Differing estrogen activities in the organic phase of air particulate matter collected during sunny and foggy weather in a Chinese city detected by a recombinant yeast bioassay

Total air suspended particles (PM 100) collected from an urban location near a traffic line in Wuhan, China, were examined for estrogen using a recombinant yeast bioassay. Wuhan, located at the central part of China, is the fourth biggest city in China with 7 million populations. Today, Wuhan has developed into the biggest city and the largest traveling center of central China, becoming one of the important bases of industry, education and research. Wuhan is right at the confluent point of Yangzi River, the third longest river in the world, and its largest distributary Hanjiang, with mountains and more than 100 takes in downtown area. Therefore, by its unique landscape, Wuhan has formed clear four seasons with relatively long winter and summer and short spring and autumn. Foggy weather usually happen in early spring. The yeast line used in this assay stably expresses human estrogen receptor-alpha. Weak but clear estrogenic activities were detected in the organic phase of crude extracts of air particle materials (APM) in both sunny and foggy weather by 0.19-0.79 mug E2/gPM(100) which were statistically significantly elevated relative to the blank control responding from 20% to 50% of the maximum E2 response, and the estrogenic activity was much higher in foggy weather than in sunny weather. The estrogenic activities in the sub-fractions from chromatographic separation of APM sampled in foggy days were also determined. The results indicated that the responses of the fractions were obviously higher than the crude extracts. Since there is no other large pollution source nearby, the estrogenic material was most likely from vehicle emissions, house heating sources and oil fumes of house cooking. The GC/MS analysis of the PM100 collected under foggy weather showed that there were many phenol derivatives, oxy-PAHs and resin acids which have been reported as environmental estrogens. These results of the analysis of estrogenic potency in sunny and foggy weather in a subtropical city of China indicate that further studies are required to investigate the actual risks for the associated health and atmospheric system. (C) 2004 Elsevier Ltd. All rights reserved.

Differential expression of thyroid-stimulating hormone beta subunit in gonads during sex reversal of orange-spotted and red-spotted groupers

We have cloned and characterized the full-length cDNA encoding thyroid-stimulating hormone beta-subunit (TSHbeta) from orange-spotted grouper Epinephelus coioides. It contains 913 nucleotides with an open reading frame encoding 146 amino acids with a 20 amino acid signal peptide. The grouper mature TSHbeta has 75, 70, 61, 59, 41, 42 and 40% identities to that of rainbow trout, Atlantic salmon, zebrafish, European eel, chicken. mouse and human, respectively. RT-PCR analysis indicated that the TSHbeta mRNA was expressed abundantly not only in pituitary but also in gonads. A more interesting finding is to reveal the differential TSHbeta expressions between the ovaries and the transitional gonads or testes in natural individuals of orange-spotted grouper and red-spotted grouper Epinephelus akaara, and in artificial sex reversal individuals of red-spotted grouper induced by MT feeding. In situ hybridization localization provided direct evidence that the TSHbeta was transcribed in the germ cells. In the growing oocytes, the TSHbeta transcripts were concentrated on the ooplasm periphery. In testicular tissues, the intensively expressed TSHbeta cells were found to be spermatogonia and spermatocytes in the spermatogenic cysts. This is the first report of a TSHbeta expressed in the gonads of any vertebrates in addition to the expected expression in the pituitary, and it expresses more transcripts in the gonads during sex reversal or testis than in the ovaries both in E. coioides and E. akaara. Importantly, the TSHbeta identification in germ cells allows us to further investigate the functional roles and the molecular mechanisms in gametogenesis of groupers, especially in sex reversal and in spermatogenesis. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

西藏齿突蟾蝌蚪冷适应策略研究

本文通过对高海拔两栖类西藏齿突蟾(Scutiger boulengeri)蝌蚪在实验室特定低温条件下的冷适应微空间行为分布的动态变化分析、温度耐受性实验及在不同适应温度的乳酸脱氢酶(LDH)同工酶的酶量与活性比较分析, 探讨了高海拔两栖类蝌蚪的部分冷适应策略。 西藏齿突蟾蝌蚪在不同温度的行为分布是一连续、动态过程，需用多种检验方法综合利用才能进行判断；在15℃, 除低海拔分布的西藏齿突蟾种群外所有实验物种蝌蚪均符合负二项分布、NeymanⅡ型分布；在10℃, 高海拔两栖类蝌蚪均符合负二项分布、NeymanⅡ型分布；在5℃、0℃低温时，高海拔两栖类不同分组的西藏齿突蟾蝌蚪的负二项分布、NeymanⅡ型分布均呈现明显差异, 这可能与高海拔两栖类蝌蚪在低温条件下通过不断地改变其行为分布方式来避免自身被冻伤有关。野外观察表明：高海拔两栖类蝌蚪常选择与流动河水相连的静水水体这种微生境中生存, 蝌蚪应对环境温度极端变化会不断改变其行为分布方式来选择最佳生存温度以避免极端高、低温对自身身体的伤害, 这种对微生境的利用能力对高海拔两栖类蝌蚪耐受极端环境温度的变化极其重要。 两栖类蝌蚪的温度耐受性实验表明不同的驯化温度可以改变西藏齿突蟾蝌蚪、两栖类仙琴水蛙蝌蚪的最适温度、逃避温度，并具有显著影响。 随着驯化温度5℃、10℃逐渐升高, 其最适温度、逃避温度也在一定范围内升高，但驯化温度对低海拔的仙琴水蛙蝌蚪的最适温度、逃避温度的改变效应大于高海拔的西藏齿突蟾蝌蚪的改变效应, 仙琴水蛙蝌蚪对温度的耐受范围、最适温度和逃避温度的ARRS值都大于西藏齿突蟾蝌蚪, 这说明仙琴水蛙蝌蚪对环境温度变化的适应能力大于西藏齿突蟾蝌蚪。 高海拔地区不同分组的两栖类蝌蚪, 在0℃适应温度时, LDH5条带的酶相对含量最高，而在5℃、10℃、15℃适应温度时，LDH5条带的酶相对含量明显都降低, 这表明酵解作用是高海拔两栖类蝌蚪的一些组织在低温﹑缺氧环境中的重要供能方式。高海拔两栖类蝌蚪同一分组的LDH总酶活性总是表现为10℃适应温度的总酶活性最高，而对低海拔的两栖类蝌蚪则是0℃适应温度的总酶活性最高, 这说明高海拔两栖类蝌蚪的LDH同工酶A、B两亚基基因活性在10℃时最高, 而低海拔两栖类蝌蚪的LDH同工酶A、B两亚基基因活性在0℃时最高。同时发现在15℃适应温度组的高海拔两栖类蝌蚪的LDH电泳图谱都有第6条带,有可能由LDH - C亚基组成, 对高海拔两栖类蝌蚪的LDH - C亚基只在15℃适应温度下才表达的机理还有待进一步的研究。 高海拔两栖类西藏齿突蟾蝌蚪通过行为分布方式的改变来选择最佳的生存温度, 这种温度选择过程与野外特定的微生境的存在密切相关, 现在由于人类对河道的不合理利用正在导致高海拔两栖类蝌蚪赖以生存的这种微生境逐渐消失, 这种微生境的消失将加速高海拔的两栖类种群数量衰退的进程。高海拔两栖类物种蝌蚪在低温（0℃）上表现出的同工酶多谱带说明，其A、B两亚基都有所表达，及其参与代谢的方式也是正常的，而低海拔两栖类物种蝌蚪只有A亚基表达的LDH5存在，因此其主要参与酵解过程，这种通过动物自身生理代谢方式的改变来适应极端环境温度条件的变化是高海拔两栖类蝌蚪能适应低温环境的重要策略。但高海拔物种的适应温度变化范围显著小于低海拔物种，对环境温度的变化适应能力有限，特别是对高温区域，因此全球气候变化可能对高海拔物种影响更为显著。 The partly cold-adaptation stratagem of the high altitude amphibian tadpole were researched in the laboratory by analyzing the high altitude amphibian tadpole of Scutiger boulengeri mainly on endpoints related to the dynamic variation of the micro-spatial behavior distribution patterns, the experiment of the temperature tolerance, and the enzyme content and activity of the lactic acid dehydrogenase(LDH) isozyme in special temperature condition. The behavior distribution of the Scutiger boulengeri tadpole is continuous and variable, but it can be figured out by multple testing ways. At 15℃, all of the experiment amphibian tadpoles behavior distribution fit both for the negative binomial distribution and NeymanⅡtype distribution except for the low altitude Scutiger boulengeri tadpoles. At 10℃, all of the high altitude amphibian tadpoles behavior distribution fit both for the negative binomial distribution and NeymanⅡtype distribution. At lower temperature, 5℃ and 0℃, the high altitude amphibian tadpoles of the Scutiger boulengeri at different groups behavior distribution fit for or don’t fit for behavior distribution respectively. It is denoted that the high altitude amphibian tadpoles probably avoid frostbiting by varying the behavior distribution patterns at low temperature condition. The high altitude amphibian tadpoles often actively select the special microhabitat which has the connected still water body and the flowing water body in the wild. It is important that tadpoles can endure the extreme temperature variety in this kind of microhabitat, because tadpoles can be better survival through select temperature condition through migrating in these kinds of microhabitats by varying their own behavior distribution patterns. Different acclimation temperature causes the significant change of preferred temperature(PT)、 avoiding temperature(AT) both in high altitude amphibian Scutiger boulengeri tadpoles and in low altitude amphibian Rana daunchina tadpoles in the temperature endurance experiment. With the acclimation temperature growing from 5℃ to 10℃. the PT and the AT of them would be uprise to some extent, but the effect of acclimation temperature on the PT and the AT of the tadpoles of Rana daunchina is more significant than the ones on the tadpoles of Scutiger boulengeri, at the same, the effects on the temperature endurance range, the ARRs of the tadpoles of Rana daunchina would be stronger than the ones on the tadpoles of Scutiger boulengeri. It is implied that the adaptation ability of tadpoles of Rana daunchina to the surroundings temperature alternation preferred to tadpoles of Scutiger boulengeri. At 0℃ acclimation temperature, the LDH5 enzyme comparative content of the high altitude amphibian tadpoles at different groups was highest, but it becomes lower at 5℃、10℃、15℃ acclimation temperature. It indicated that the alcoholysis role was the important ways of applying energy for special tissue of the high altitude amphibian tadpoles in low-temperature and low-oxygen condition. The total enzyme activity of the LDH of the high altitude amphibian tadpoles in the same group always keeps the highest at 10℃ acclimation temperature, but the low altitude amphibian tadpoles’ was maximum at 0℃. It was denoted that the gene activity of LDH -A and LDH – B submit was highest at 10℃ acclimation temperature for the high altitude amphibian tadpoles, but the low altitude amphibian tadpoles’ was maximum at 0℃. Meanwhile, the LDH electrophoretogram of the high altitude amphibian tadpoles always composed of 6 stripes at 15℃ acclimation temperature，the extra stripe probably was composed by LDH-C submit。It is unknown why LDH-C expresses only under high temperature。. The high altitude amphibian tadpoles can select the most optimal temperature by changing their behavior distribution patterns ceaselessly, but this course of selecting the most suitable temperature correlated with the special microhabitat in the wild closely. Nowadays, this kind of microhabitat which the high altitude amphibian tadpoles rely on are lossing gradually for human being exploit the riverway unreasonably. The disappearing of the microhabitat would accelerate the decline of the high altitude amphibian population. Compare to one band of LDH5, which only composed by the LDH-A submit, presents in the low altitude amphibian at 0℃, the five bands which composed by the LDH-A and LDH-B are checked out, this means the species which occurred in the highland is more adaptable to the low temperature. It is an important stratagem for the high altitude amphibian tadpoles adapt to the limited low temperature depends on the animal energy metabolism change.However, this kind of adaption is restricted, the adaption range to the temperature is much norrow in the high altitude amphibian than in the low one, especially for the high temperature side. The global climate change will be more serious for the high altitude species.

Attenuation of Edwardsiella tarda Virulence by Small Peptides That Interfere with LuxS/Autoinducer Type 2 Quorum Sensing

Edwardsiella tarda is a gram-negative pathogen with a broad host range that includes humans, animals, and fish. Recent studies have shown that the LuxS/autoinducer type 2 (AI-2) quorum sensing system is involved in the virulence of E. tarda. In the present study, it was found that the E. tarda LuxS mutants bearing deletions of the catalytic site (C site) and the tyrosine kinase phosphorylation site, respectively, are functionally inactive and that these dysfunctional mutants can interfere with the activity of the wild-type LuxS. Two small peptides, 5411 and 5906, which share sequence identities with the C site of LuxS, were identified. 5411 and 5906 proved to be inhibitors of AI-2 activity and could vitiate the infectivity of the pathogenic E. tarda strain TX1. The inhibitory effect of 5411 and 5906 on AI-2 activity is exerted on LuxS, with which these peptides specifically interact. The expression of 5411 and 5906 in TX1 has multiple effects (altering biofilm production and the expression of certain virulence-associated genes), which are similar to those caused by interruption of luxS expression. Further study found that it is very likely that 5411 and 5906 can be released from the strains expressing them and, should TX1 be in the vicinity, captured by TX1. Based on this observation, a constitutive 5411 producer (Pseudomonas sp. strain FP3/pT5411) was constructed in the form of a fish commensal isolate that expresses 5411 from a plasmid source. The presence of FP3/pT5411 in fish attenuates the virulence of TX1. Finally, it was demonstrated that fish expressing 5411 directly from tissues exhibit enhanced resistance against TX1 infection.

Attenuation of Edwardsiella tarda Virulence by Small Peptides That Interfere with LuxS/Autoinducer Type 2 Quorum Sensing

Edwardsiella tarda is a gram-negative pathogen with a broad host range that includes humans, animals, and fish. Recent studies have shown that the LuxS/autoinducer type 2 (AI-2) quorum sensing system is involved in the virulence of E. tarda. In the present study, it was found that the E. tarda LuxS mutants bearing deletions of the catalytic site (C site) and the tyrosine kinase phosphorylation site, respectively, are functionally inactive and that these dysfunctional mutants can interfere with the activity of the wild-type LuxS. Two small peptides, 5411 and 5906, which share sequence identities with the C site of LuxS, were identified. 5411 and 5906 proved to be inhibitors of AI-2 activity and could vitiate the infectivity of the pathogenic E. tarda strain TX1. The inhibitory effect of 5411 and 5906 on AI-2 activity is exerted on LuxS, with which these peptides specifically interact. The expression of 5411 and 5906 in TX1 has multiple effects (altering biofilm production and the expression of certain virulence-associated genes), which are similar to those caused by interruption of luxS expression. Further study found that it is very likely that 5411 and 5906 can be released from the strains expressing them and, should TX1 be in the vicinity, captured by TX1. Based on this observation, a constitutive 5411 producer (Pseudomonas sp. strain FP3/pT5411) was constructed in the form of a fish commensal isolate that expresses 5411 from a plasmid source. The presence of FP3/pT5411 in fish attenuates the virulence of TX1. Finally, it was demonstrated that fish expressing 5411 directly from tissues exhibit enhanced resistance against TX1 infection.

Construction and evaluation of DNA vaccines encoding Edwardsiella tarda antigens

Edwardsiella tarda is an opportunistic pathogen that can infect humans, animal, and fish. Two E. tarda antigens, Eta6 and FliC, which are homologues to an ecotin precursor and the FliC flagellin, respectively, were identified by in vivo-induced antigen technology from a pathogenic E. tarda strain isolated from diseased fish. When used as a subunit vaccine, purified recombinant Eta6 was moderately protective against lethal challenge of E. tarda in a Japanese flounder model, whereas purified recombinant FliC showed no apparent immunciprotectivity. Similarly, DNA vaccines based on eta6 and fliC in the form of plasmids pEta6 and pFliC induced, respectively, moderate and marginal protection against E. tarda infection. To improve the vaccine efficacy of eta6, a chimeric DNA vaccine, pCE6, was constructed, which encodes Eta6 fused in-frame to FliC. pCE6 was found to induce significantly higher level of protection than pEta6. Likewise, another chimeric DNA vaccine, pCE18, which expresses FliC fused to a previously identified E. tarda antigen Et18, elicited significantly stronger protective immunity than the DNA vaccine based on et18 alone. Fish immunized with pEta6 and pCE6 produced specific serum antibodies and exhibited significantly enhanced expression of the genes encoding elements that are involved in both innate and adaptive immune responses. Furthermore, the induction magnitudes of most of these genes were significantly higher in pCE6-vaccinated fish than in pEta6-vaccinated fish. (C) 2009 Elsevier Ltd. All rights reserved.

Identification and immunoprotective analysis of an in vivo-induced Edwardsiella tarda antigen

Edwardsiella tarda is a severe aquaculture pathogen that can infect many important fish species cultured worldwide. The aim of this study was to evaluate the vaccine potential of an E. tarda antigen, Eta21, which was identified from a pathogenic E. tarda strain via the method of in vivo-induced antigen technology (IVIAT). Eta21 is 510-amino acid in length and shares similar to 58% sequence identity with a putative peptidase of several bacterial species. eta21 was subcloned into Escherichia colt, and recombinant Eta21 was purified as a histidine-tagged protein. When used as a subunit vaccine, purified recombinant Eta21 was effective against lethal E. tarda challenge in a Japanese flounder model. In order to improve the immunoprotective efficacy of Eta21, the chimera AgaV-Eta21 was constructed, which consists of Eta21 fused in-frame to the secretion domain of AgaV, an extracellular beta-agarase. E. coli DH5 alpha harboring plasmid pTAET21, which constitutively expresses agaV-eta21, was able to produce and secret AgaV-Eta21 into the extracellular milieu. Vaccination of Japanese flounder with live DH5 alpha/pTAET21 elicited immunoprotection that is significantly higher in level than that induced by vaccination with purified recombinant Eta21. Vaccination with DH5 alpha/pTAET21 and recombinant Eta21 both induced the production of specific serum antibodies at four to eight weeks post-vaccination. Taken together, these results demonstrate that Eta21, especially that delivered by DH5 alpha/pTAET21, is an effective vaccine candidate against E. tarda infection. (C) 2009 Elsevier Ltd. All rights reserved.

Molecular cloning and characterisation of a pattern recognition protein, lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) from Chinese shrimp Fenneropenaeus chinensis

A pattern recognition protein (PRP), lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) cDNA was cloned from the haemocyte of Chinese shrimp Fenneropenaeus chinensis by the techniques of homology cloning and RACE. Analysis of nucleotide sequence revealed that the full-length cDNA of 1,275 bp has an open reading frame of 1,098 bp encoding a protein of 366 amino acids including a 17 amino acid signal peptide. Sequence comparison of the deduced amino acid sequence of F. chinensis LGBP showed a high identity of 94%, 90%, 87%, 72% and 63% with Penaeus monodon BGBP, Litopenaeus stylirostris LGBP, Marsupenaeu japonicus BGBP, Homarus gammarus BGBP and Pacifastacus leniusculus LGBP, respectively. The calculated molecular mass of the mature protein is 39,857 Da with a deduced pI of 4.39. Two putative integrin binding motifs, RGD (Arg-Gly-Asp) and a potential recognition motif for beta-1,3-linkage of polysaccharides were observed in LGBP sequence. RT-PCR analysis showed that LGBP gene expresses in haemocyte and hepatopancreas only, but not in other tissues. Capillary electrophoresis RT-PCR method was used to quantify the variation of mRNA transcription level during artificial infection with heat-killed Vibrio anguillarum and Staphylococcus aureusin. A significant enhancement of LGBP transcription was appeared at 6 h post-injection in response to bacterial infection. These results have provided useful information to understand the function of LGBP in shrimp.

Immunoprotective analysis of VhhP2, a Vibrio harveyi vaccine candidate

VhhP2 is an Outer membrane protein identified in a pathogenic Vibrio harveyi strain, T4, isolated from diseased fish. When used as a Subunit Vaccine, purified recombinant VhhP2 affords high level of protection upon Japanese flounder against V harveyi challenge. Vaccination with VhhP2 induced the expression of a number of immune-related genes, especially those encoding immunoglobulin M (IgM) and major histocompatibility complex (MHC) II alpha. A VhhP2 surface display system, in the form of the fish commensal strain FIR harboring the vhhP2-expressing plasmid pJVP, was constructed. PF3/pJVP is able to produce and present recombinant VhhP2 on cell surface. Vaccination of fish with live PF3/pJVP via intraperitoneal injection elicited Strong immunoprotection. Vaccination of fish orally with live PF3/pJVP embedded in alginate microspheres also induced effective immunoprotection. In addition, a VhhP2-based surface display system was created, in which VhhP2 serves as a carrier for the Surface delivery of a heterologous Edwardsiella tarda immunogen, Et18, that is fused in-frame to VhhP2. DH5 alpha/pJVP18, which expresses and surface-displays the VhhP2-Et18 chimera, proved to be an effective vaccine that call protect fish against infections by V. harveyi and E. tarda to the extents comparable to those produced by vaccination with purified recombinant VhhP2 and Et18, respectively. These data suggest that VhhP2 may be applied as a vaccine and a vaccine carrier against infections by V. harveyi and other pathogens such as F. tarda. (C) 2009 Elsevier Ltd. All rights reserved.

Construction of an attenuated Pseudomonas fluorescens strain and evaluation of its potential as a cross-protective vaccine

Ferric uptake regulator (Fur) is a global transcription regulator that is ubiquitous to Gram-negative bacteria and regulates diverse biological processes, including iron uptake, cellular metabolism, stress response, and production of virulence determinants. As a result, for many pathogenic bacteria, Fur plays a crucial role in the course of infection and disease development. In this study, the fur gene was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased Japanese flounder cultured in a local farm. TSS Fur can partially complement the defective phenotype of an Escherichia coli fur mutant. A TSS fur null mutant, TFM, was constructed. Compared to TSS, TFM exhibits reduced growth ability, aberrant production of outer membrane proteins, decreased resistance against host serum bactericidal activity, impaired ability to disseminate in host blood and tissues, and drastic attenuation in overall bacterial virulence in a Japanese flounder infection model. When used as a live vaccine administered via the injection, immersion, and oral routes, TFM affords high levels of protection upon Japanese flounder against not only P.fluorescens infection but also Aeromonas hydrophila infection. Furthermore, a plasmid, pJAQ, was constructed, which expresses the coding element of the Vibrio harveyi antigen AgaV-DegQ. TFM harboring pJAQ can secret AgaV-DegQ into the extracellular milieu. Vaccination of Japanese flounder with live TFM/pJAQ elicited strong immunoprotection against both V. harveyi and A. hydrophila infections. (C) 2009 Elsevier Ltd. All rights reserved.