Identification of an Edwardsiella tarda surface antigen and analysis of its immunoprotective potential as a purified recombinant subunit vaccine and a surface-anchored subunit vaccine expressed by a fish commensal strain

Edwardsiella tarda is the etiological agent of edwardsiellosis, a systematic disease that affects a wide range of marine and freshwater fish cultured worldwide. In order to identify E. tarda antigens with vaccine potential, we in this study conducted a systematic search for E. tarda proteins with secretion capacity. One of the proteins thus identified was Esa1, which contains 795 amino acid residues and shares extensive overall sequence identities with the D15-like surface antigens of several bacterial species. In silico analyses indicated that Esa1 localizes to outer membrane and possesses domain structures that are conserved among bacterial surface antigens. The vaccine potential of purified recombinant Esa1 was examined in a Japanese flounder (Paralichthys olivaceus) model, which showed that fish vaccinated with Esa1 exhibited a high level of survival and produced specific serum antibodies. Passive immunization of naive fish with antisera raised against Esa1 resulted in significant protection against E. tarda challenge. Taking advantage of the secretion capacity of Esa1 and the natural gut-colonization ability of a fish commensal strain, we constructed an Esa1-expressing recombinant strain, FP3/pJsa1. Western immunoblot and agglutination analyses showed that FP3/pJsa1 produces outer membrane-localized Esa1 and forms aggregates in the presence of anti-Esa1 antibodies. Vaccination analyses showed that FP3/pJsa1 as an intraperitoneal injection vaccine and an oral vaccine embedded in alginate microspheres produced relative percent survival rates of 79% and 52%, respectively, under severe challenging conditions that resulted in 92-96% mortality in control fish. Further analyses showed that following oral vaccination, FP3/pJsa1 was able to colonize in the gut but unable to disseminate into other tissues. Together these results indicate that Esa1 is a protective immunogen and an effective oral vaccine when delivered by FP3/pJsa1 as a surface-anchored antigen. (c) 2010 Elsevier Ltd. All rights reserved.

Isolation and analysis of the vaccine potential of an attenuated Edwardsiella tarda strain

Edwardsiella tarda is an important aquaculture pathogen that can infect a wide range of marine and freshwater fish worldwide. In this study, a modified E. tarda strain, TX5RM, was selected by multiple passages of the pathogenic E. tarda strain TX5 on growth medium containing the antibiotic rifampicin. Compared to the wild type strain, the rifampicin-resistant mutant TX5RM (i) shows drastically increased median lethal dose and reduced capacity to disseminate in and colonize fish tissues and blood; (ii) exhibits slower growth rates when cultured in rich medium or under conditions of iron depletion; and (iii) differs in the production profile of whole-cell proteins. The immunoprotective potential of TX5RM was examined in a Japanese flounder (Paralichthys olivaceus) model as a vaccine delivered via intraperitoneal injection, oral feeding, bath immersion, and oral feeding plus immersion. All the vaccination trials, except those of injection, were performed with a booster at 3-week after the first vaccination. The results showed that TX5RM administered via all four approaches produced significant protection, with the highest protection levels observed with TX5RM administered via oral feeding plus immersion, which were, in terms of relative percent of survival (RPS), 80.6% and 69.4% at 5- and 8-week post-vaccination, respectively. Comparable levels of specific serum antibody production were induced by TX5RM-vaccinated via different routes. Microbiological analyses showed that TX5RM was recovered from the gut, liver, and spleen of the fish at 1-10 days post-oral vaccination and from the spleen, liver, kidney, and blood of the fish at 1-14 days post-immersion vaccination. Taken together, these results indicate that TX5RM is an attenuated E. tarda strain with good vaccine potential and that a combination of oral and immersion vaccinations may be a good choice for the administration of live attenuated vaccines. (C) 2010 Elsevier Ltd. All rights reserved.

Evaluation of the pyrolytic and kinetic characteristics of Enteromorpha prolifera as a source of renewable bio-fuel from the Yellow Sea of China

The pyrolytic and kinetic characteristics of Enteromorpha prolifera from the Yellow Sea were evaluated at heating rates of 10, 20 and 50 degrees C min(-1), respectively. The results indicated that three stages appeared during pyrolysis; dehydration, primary devolatilization and residual decomposition. Differences in the heating rates resulted in considerable differences in the pyrolysis of E. prolifera. Specifically, the increase of heating rates resulted in shifting of the initial temperature, peak temperature and the maximum weight loss to a higher value. The average activation energy of E. prolifera was 228.1 kJ mol(-1), the pre-exponential factors ranged from 49.93 to 63.29 and the reaction orders ranged from 2.2 to 3.7. In addition, there were kinetic compensation effects between the pre-exponential factors and the activation energy. Finally, the minimum activation energy was obtained when a heating rate of 20 degrees C min(-1) was used. (C) 2009 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Identification and analysis of a CpG motif that protects turbot (Scophthalmus maximus) against bacterial challenge and enhances vaccine-induced specific immunity

Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs in certain contexts are known to be immunostimulatory in vertebrate systems. CpG ODNs with immune effects have been identified for many fish species but, to our knowledge, not for turbot. In this study, a turbot-effective CpG ODN, ODN 205, was identified and a plasmid, pCN5, was constructed which contains the CpG motif of ODN 205. When administered into turbot via intraperitoneal (i.p.) injection, both ODN 205 and pCN5 could (i) inhibit bacterial dissemination in blood in dose and time dependent manners, and (ii) protect against lethal bacterial challenge. Immunological analyses showed that in vitro treatment with ODN 205 stimulated peripheral blood leukocyte proliferation, while i.p. injection with ODN 205 enhanced the respiratory burst activity, chemiluminescence response, and acid phosphatase activity of turbot head kidney macrophages. pCN5 treatment-induced immune responses similar to those induced by ODN 205 treatment except that pCN5 could also enhance serum bactericidal activity in a calcium-independent manner. To examine whether ODN 205 and pCN5 had any effect on specific immunity, ODN 205 and pCN5 were co-administered into turbot with a Vibrio harveyi subunit vaccine, DegQ. The results showed that pCN5, but not ODN 205, significantly increased the immunoprotective efficacy of DegQ and enhanced the production of specific serum antibodies in the vaccinated fish. Further analysis indicated that vaccination with DegQ in the presence of pCN5 upregulated the expression of the genes encoding MHC class II alpha, IgM, Mx, and IL-8 receptor. Taken together, these results demonstrate that ODN 205 and pCN5 can stimulate the immune system of turbot and induce protection against bacterial challenge. In addition, pCN5 also possesses adjuvant property and can potentiate vaccine-induced specific immunity. (C) 2010 Elsevier Ltd. All rights reserved.

Construction and analysis of an experimental Streptococcus iniae DNA vaccine

Streptococcus iniae is a severe aquaculture pathogen that can also infect humans and animal. A putative secretory antigen, Slat 0, was identified from a pathogenic S. iniae strain by in vivo-induced antigen technology. Using turbot as an animal model, the immunoprotective effect of Sia10 was examined as a DNA vaccine in the form of plasmid pSia10, which expresses sia10 under the cytomegalovirus immediate-early promoter. In fish vaccinated with pSia10, transcription of sia10 was detected in muscle, liver, spleen, and kidney at 7, 14, 21, 28, 35, 42, and 49 days post-vaccination. In addition, production of Sia10 protein was also detected in the muscle tissues of pSia10-vaccinated fish. Fish vaccinated with pSia10 exhibited a relative percent survival (RPS) of 73.9% and 92.3%, respectively, when challenged with high and low doses (producing a cumulative mortality of 92% and 52%, respectively, in the control groups) of S. iniae. Immunological and transcriptional analyses showed that vaccination with pSia10(i) induced much stronger chemiluminescence response and significantly higher levels of nitric oxide production and acid phosphatase activity in head kidney macrophages; (ii) caused the production of specific serum antibodies, which afforded apparent immunoprotection when transferred passively into naive fish; and (iii) upregulated the expression of the genes encoding proteins that are possibly involved in both innate and adaptive immune responses. Taken together, these results indicated that pSia10 is an effective vaccine candidate and may be used in the control of S. iniae infection in aquaculture. (C) 2010 Elsevier Ltd. All rights reserved.

The dominant Ulva strain of the 2008 green algal bloom in the Yellow Sea was not detected in the coastal waters of Qingdao in the following winter

The region of Qingdao, China, experienced the world's largest green tide from May to July 2008. More than one million tons of fresh algal biomass of the green alga Ulva prolifera was harvested, while more was suspected to have sunk to the bottom. The original source of this seaweed was suspected to be from the south as revealed by satellite images. The floating biomass drifted with the water current northward and flourished in nearshore waters around Qingdao. However, direct biological evidence for "seed" source is lacking. It is still unclear whether this alga could survive the Qingdao local coastal environment and pose future danger of potential blooming. Systematic and seasonal sampling of waters in the intertidal zone at six collection sites along the Qingdao coast was conducted from December 2008 to April 2009. Forty-eight water samples were analyzed. From these, nine different morphotypes of Ulva were grown in the laboratory under standard temperature and light regimes. Growth of Ulva was observed in all water samples. However, molecular phylogenetic analyses revealed that the dominant U. prolifera strain of the 2008 bloom was absent in all the water-derived cultures during the sampling period. These results provide evidence that the dominant bloom-forming alga was unlikely able to survive the coastal waters (the minimal surface water temperature in February is 2A degrees C) in winter conditions in Qingdao, even though all the sampling locations were heavily covered by this alga in June 2008.

Ulva and Enteromorpha (Ulvaceae, Chlorophyta) from two sides of the Yellow Sea: analysis of nuclear rDNA ITS and plastid rbcL sequence data

Ulvacean green seaweeds are common worldwide; they formed massive green tides in the Yellow Sea in recent years, which caused marine ecological problems as well as a social issue. We investigated two major genera of the Ulvaceae, Ulva and Enteromorpha, and collected the plastid rbcL and nuclear ITS sequences of specimens of the genera in two sides of the Yellow Sea and analyzed them. Phylogenetic trees of rbcL data show the occurrence of five species of Enteromorpha (E. compressa, E. flexuosa, E. intestinalis, E. linza and E. prolifera) and three species of Ulva (U. pertusa, U. rigida and U. ohnoi). However, we found U. ohnoi, which is known as a subtropical to tropical species, at two sites on Jeju Island, Korea. Four ribotypes in partial sequences of 5.8S rDNA and ITS2 from E. compressa were also found. Ribotype network analysis revealed that the common ribotype, occurring in China, Korea and Europe, is connected with ribotypes from Europe and China/Japan. Although samples of the same species were collected from both sides of the Yellow Sea, intraspecific genetic polymorphism of each species was low among samples collected worldwide.

Quantitatively distinguishing sediments from the Yangtze River and the Yellow River using delta Eu-N-I REEs pound plot

Sediment samples were collected from the lower channel of the Yangtze River and the Yellow River and the contents of rare earth elements (REEs) were measured. In addition, some historical REEs data were collected from published literatures. Based on the delta Eu-N-I REEs pound plot, a clear boundary was found between the sediments from the two rivers. The boundary can be described as an orthogonal polynomial equation by ordinary linear regression with sediments from the Yangtze River located above the curve and sediments from the Yellow River located below the curve. To validate this method, the REEs contents of sediments collected from the estuaries of the Yangtze River and the Yellow River were measured. In addition, the REEs data of sediment Core 255 from the Yangtze River and Core YA01 from the Yellow River were collected. Results show that the samples from the Yangtze River estuary and Core 255 almost are above the curve and most samples from the Yellow River estuary and Core YA01 are below the curve in the delta Eu-N-I REEs pound plot. The plot and the regression equation can be used to distinguish sediments from the Yangtze River and the Yellow River intuitively and quantitatively, and to trace the sediment provenance of the eastern seas of China. The difference between the sediments from two rivers in the delta Eu-N-I REEs pound plot is caused by different mineral compositions and regional climate patterns of the source areas. The relationship between delta Eu-N and I REEs pound is changed little during the transport from the source area to the river, and from river to the sea. Thus the original information on mineral compositions and climate of the source area was preserved.

Formation and evolution of the modern warm current system in the East China Sea and the Yellow Sea since the last deglaciation

To reconstruct the formation and evolution process of the warm current system within the East China Sea (ECS) and the Yellow Sea (YS) since the last deglaciation, the paleoceangraphic records in core DGKS9603, core CSH1 and core YSDP102, which were retrieved from the mainstream of the Kuroshio Current (KC), the edge of the modern Tsushima Warm Current (TWC) and muddy region under cold waters accreted with the Yellow Sea Warm Current (YSWC) respectively, were synthetically analyzed. The results indicate that the formation and evolution of the modern warm current system in the ECS and the YS has been accompanied by the development of the KC and impulse rising of the sea level since the last deglaciation. The influence of the KC on the Okinawa Trough had enhanced since 16 cal kyr BP, and synchronously the modern TWC began to develop with the rising of sea level and finally formed at about 8.5 cal kyr BP. The KC had experienced two weakening process during the Heinrich event 1 and the Younger Drays event from 16 to 8.5 cal kyr BP. The period of 7-6 cal kyr BP was the strongest stage of the KC and the TWC since the last deglaciation. The YSWC has appeared at about 6.4 cal kyr BP. Thus, the warm current system of the ECS and the YS has ultimately formed. The weakness of the KC, indicated by the occurrence of Pulleniatina minimum event (PME) during the period from 5.3 to 2.8 cal kyr BP, caused the main stream of the TWC to shift eastward to the Pacific Ocean around about 3 cal kyr BP. The process resulted in the intruding of continent shelf cold water mass with rich nutrients. Synchronously, the strength of the YSWC was relatively weak and the related cold water body was active at the early-mid stage of its appearance against the PME background, which resulted in the quick formation of muddy deposit system in the southeastern YS. The strength of the warm current system in the ECS and the YS has enhanced evidently, and approached to the modern condition gradually since 3 cal kyr BP.

Rare earth elements in bottom sediments of major rivers around the Yellow Sea: implications for sediment provenance

Rare earth elements (REEs) of 91 fine-grained bottom sediment samples from five major rivers in Korea (the Han, Keum, and Yeongsan) and China (the Changjiang and Huanghe) were studied to investigate their potential as source indicator for Yellow Sea shelf sediments, this being the first synthetic report on REE trends for bottom sediments of these rivers. The results show distinct differences in REE contents and their upper continental crust (UCC)-normalized patterns: compared to heavy rare earth elements (HREEs), light rare earth elements (LREEs) are highly enriched in Korean river sediments, in contrast to Chinese river sediments that have a characteristic positive Eu anomaly. This phenomenon is observed also in primary source rocks within the river catchments. This suggests that source rock composition is the primary control on the REE signatures of these river sediments, due largely to variations in the levels of chlorite and monazite, which are more abundant in Korean bottom river sediments. Systematic variations in I LREE pound/I HREE pound ratios, and in (La/Yb)-(Gd/Yb)(UCC) but also (La/Lu)-(La/Y)(UCC) and (La/Y)-(Gd/Lu)(UCC) relations have the greatest discriminatory power. These findings are consistent with, but considerably expand on the limited datasets available to date for suspended sediments. Evidently, the REE fingerprints of these river sediments can serve as a useful diagnostic tool for tracing the provenance of sediments in the Yellow Sea, and for reconstructing their dispersal patterns and the circulation system of the modern shelf, as well as the paleoenvironmental record of this and adjoining marginal seas.

Sporopollen analysis of Core B10 in the southern Yellow Sea and the reflected characteristics of climate changes

Eight sporopollen zones have been divided based on the results of high-resolution sporopollen analysis of Core B10 in the southern Yellow Sea. Based on the results along with C-14 datings and the subbottom profiling data, climatic and environmental changes since the last stage of late Pleistocene are discussed. The main conclusions are drawn as follows: (1) the vegetation evolved in the process of coniferous forest-grassland containing broad-leaved treesconiferous and broad-leaved mixed forest --> coniferous and broad-leaved mixed forest-grassland prevailed by coniferous trees --> coniferous and broad-leaved mixed forest-grassland containing evergreen broad-leaved trees- coniferous and broad-leaved mixed forest-grassland prevailed by broad-leaved trees-deciduous broad-leaved forest-meadow containing evergreen broad-leaved trees- coniferous and broadleaved mixed forest-grassland prevailed by broad-leaved trees- coniferous and broad-leaved mixed forest containing evergreen broad-leaved trees; (2) eight stages of climate changes are identified as the cold and dry stage, the temperate and wet stage, the cold and dry stage, the warm and dry stage, the temperate and wet stage, the hot and dry stage, the temperate and dry stage, then the warm and dry stage in turn; (3) the sedimentary environment developed from land, to littoral zone, to land again, then to shore-neritic zone; and (4) the Yellow Sea Warm Current formed during early-Holocene rather than Atlantic stage.

Modern sedimentary environments and dynamic depositional systems in the southern Yellow Sea

Based on analyses of more than 600 surface sediment samples together with large amounts of previous sedimentologic and hydrologic data, the characteristics of modern sedimentary environments and dynamic depositional systems in the southern Yellow Sea (SYS) are expounded, and the controversial formation mechanism of muddy sediments is also discussed. The southern Yellow Sea shelf can be divided into low-energy sedimentary environment and high-energy sedimentary environment; the low-energy sedimentary environment can be further divided into cyclonic and anticyclonic ones, and the high-energy environment is subdivided into high-energy depositional and eroded environments. In the shelf low-energy environments, there developed muddy depositional system. In the central part of the southern Yellow Sea, there deposited the cold eddy sediments under the actions of a meso-scale cyclonic eddy (cold eddy), and in the southeast of the southern Yellow Sea, an anticyclonic eddy muddy depositional system (warm eddy sediment) was formed. These two types of sediments showed evident differences in grain size, sedimentation rate, sediment thickness and mineralogical characteristics. The high-energy environments were covered with sandy sediments on seabed; they appeared mainly in the west, south and northeast of the southern Yellow Sea. In the high-energy eroded environment, large amounts of sandstone gravels were distributed on seabed. In the high-energy depositional environment, the originally deposited fine materials (including clay and fine silt) were gradually re-suspended and then transported to a low-energy area to deposit again. In this paper, the sedimentation model of cyclonic and anticyclonic types of muddy sediments is established, and a systematic interpretation for the formation cause of muddy depositional systems in the southern Yellow Sea is given.

Sediment provenance and province of the southern Yellow Sea: Evidence from light mineral

The distribution for percent content of light mineral is divided in detail to emphasize distributional trends of higher and lower contents by using 222 samples of light mineral in the southern Yellow Sea. 5 mineral provinces are divided, and they are I-north mineral province of the southern Yellow Sea, the sediment dominantly derived from the Yellow River; II-mixed mineral province, the sediment derived from both the Yellow River and Yangtze River; III-middle mineral province, the sediment derived mainly from the Yellow River and a part of sediment derived from Yangtze River; IV-province east of Yangtze River mouth, the sediment derived dominantly from Yangtze River; and V south mineral province, sediment was affected by relict sediment and modern sediment of Yangtze River. In this paper, the assemblage of dominant mineral and diagnostic mineral for the five provinces are discerned.

Clay mineral distributions in the southern Yellow Sea and their significance

To better understand the characteristics of the clay minerals in the southern Yellow Sea, the X-ray quantitative determinations have been carried out for the surface samples obtained from the Yellow Sea. With newly compiled clay mineral synoptic maps, the depositional processes were described for four main clay minerals (illite, chlorite, kaolinite and smectite). The analysis shows that most clay minerals are of terrigenous source with the Huanghe River acting as the major sediment supplier. Besides, the source of muddy sediments in the Yellow Sea was also discussed. As for the central Yellow Sea mud (CYSM), the sediments in its northern part mainly come from the Huanghe River, and those in the rest are of multi-origin. Very similarly, a large amount of sediments in the northern part of the southeastern Yellow Sea Mud (SEYSM) derive from the Keum River and Yeong-san River, while those in the southern part are of multi-origin.

The Shandong mud wedge and post-glacial sediment accumulation in the Yellow Sea

Two well-defined deltaic sequences in the Bohai Sea and in the South Yellow Sea represent post-glacial accumulation of Yellow River-derived sediments. Another prominent depocenter on this epicontinental shelf, a pronounced clinoform in the North Yellow Sea, wraps around the northeastern and southeastern end of the Shandong Peninsula, extending into the South Yellow Sea. This Shandong mud wedge is 20 to 40 m thick and contains an estimated 300 km(3) of sediment. Radiocarbon dating, shallow seismic profiles, and regional sea-level history suggest that the mud wedge formed when the rate of post-glacial sea-level rise slackened and the summer monsoon intensified, at about 11 ka. Geomorphic configuration and mineralogical data indicate that present-day sediment deposited on the Shandong mud wedge comes not only from the Yellow River but also from coastal erosion and local rivers. Basin-wide circulation in the North Yellow Sea may transport and redistribute fine sediments into and out of the mud wedge.