Skin Ulceration Syndrome Research Articles

Screening and characteristic analysis of Bacillus velezensis from sea cucumber ( Apostichopus japonicus ) ponds

PDF HTML阅读 XML下载 导出引用 引用提醒 刺参养殖池塘一株贝莱斯芽孢杆菌的分离及其生理特性 DOI: 作者: 作者单位: 1. 上海海洋大学 水产与生命学院, 上海 201306;2. 中国水产科学研究院黄海水产研究所, 青岛海洋科学与技术国家实验室, 海洋渔业科学与食物产出过程功能实验室, 山东 青岛 266071;3. 山东海跃水产科技有限公司, 山东 东营 257500 作者简介: 王成强(1988-),男,研究实习员,主要从事水产动物营养与饲料研究.E-mail:chengqiangwang@126.com 通讯作者: 中图分类号: S947 基金项目: 山东省农业良种工程重大课题；山东省自主创新与成果转化专项（2014ZZCX06205）；中央级公益性科研院所基本科研业务费专项资金项目（20603022016008）. Screening and characteristic analysis of Bacillus velezensis from sea cucumber (Apostichopus japonicus) ponds Author: Affiliation: 1. Shanghai Ocean University, Shanghai 201306, China;2. College of Fesheries and Life Science, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences;Qingdao National Laboratory for Marine Science and Technology, Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao 266071, China;3. Shandong Haiyue Aquatic Science and Technology Co., Ltd, Dongying 257500, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:2014年5月，在东营的刺参（）养殖池塘环境中对水样和底泥样品进行了细菌分离和益生菌的筛选，共获得66株可培养细菌。以刺参“腐皮综合征”主要致病菌为指示菌进行拮抗作用实验，使用选择培养基对菌株的产淀粉酶和蛋白酶能力进行测定，筛选获得一株潜在益生菌株——DY-6，并进行了生理生化实验、16S rDNA基因序列分析、菌株生长特性及其对刺参的安全性研究。结果表明，DY-6对其指示菌假交替单胞菌（Vibrio parahaemolyticus）和溶藻弧菌（）均有良好的抑制作用，抑菌圈直径分别达到24 mm、22 mm、27 mm和37 mm，对淀粉和蛋白培养基的水解直径分别为28 mm和20 mm。利用该菌对刺参进行高浓度胁迫实验测试其安全性，实验期间浸浴组和投喂组刺参状态良好，没有发病和死亡现象。利用细菌生理生化和16S rDNA基因序列分析表明，DY-6与贝莱斯芽孢杆菌（ CP015911）的相似性为99%。因此，初步鉴定该菌株为贝莱斯芽孢杆菌。同时，将该菌株与商品化微生态制剂产品中分离的4株益生菌进行了比较，DY-6在温度20~35℃、盐度0~35范围内生长较快，优于上述4株菌，4 h进入对数生长期，10 h达到生长高峰。筛选的贝莱斯芽孢杆菌具有良好的抑菌能力，生长速度快，并且具有广温广盐的优点，不仅能够降低疾病发生率，还可更好地适应黄河口地区夏季水温偏高、盐度波动大的特点，由此具有良好的益生菌商业化开发潜力。 Abstract:To screen local probiotic strains, 66 cultivable bacterial strains were isolated from water and sediment samples from cultured ponds of sea cucumber () in Dongying in May 2014. Bacterial antagonistic experiments were conducted using V. parahaemolyticus and , which are main pathogenic bacteria of ‘skin ulcer syndrome’ for . In addition, selective medium was used to test the amylase production and protease activities of the isolated strains and to isolate a potential probiotic strain, DY-6. Then, its physiological-biochemical experiments and similarity analysis of 16s rDNA sequences as well as the growth characteristics of DY-6 experiments have been conducted. The safety experiment for the sea cucumber was also proceed. The DY-6 strain was successful in inhibiting V. alginolyticus, as indicated by inhibition zone diameters of 24, 22, 27, and 37 mm, respectively. Meanwhile, the hydrolysis zone diameters of amylase and protease were 28 and 20 mm, respectively. A safety experiment was designed to test the safety of DY-6 for sea cucumber. During the experiment, all sea cucumbers were in good conditions without falling ill. Based on physiological and biochemical experiments and 16s rDNA sequence analysis, strain DY-6 was determined to be 99% similar to B. velezensis. When compared to four bacterial strains isolated from commercial probiotic products, DY-6 exhibited superior growth at 20-35℃ and under salinities of 0 to 35. Moreover, DY-6 came into the logarithmic growth phase after 4 h of cultivation and reached growth peak after 10 h. In summary, strain DY-6 exhibited superb inhibition of sea cucumber pathogens, rapid growth, and both euryhalinous and euryhaline features. Therefore, DY-6 could be used to reduce the occurrence of sea cucumber disease and is also suitable for use at high temperatures during summer and in fluctuation salinity levels, as in the Yellow River Estuary. Thus, strain DY-6 has potential for commercial development. 参考文献 相似文献 引证文献

Characterization of a metalloprotease involved in Vibrio splendidus infection in the sea cucumber, Apostichopus japonicus

Vibrio splendidus is an important aquatic pathogen that infects a broad range of hosts, leading to typical symptoms of “skin ulceration syndrome” in the sea cucumber Apostichopus japonicus. However, there are few reports on the virulence factors and pathogenic mechanism of V. splendidus. In the present study, V. splendidus could survive in the coelomic fluid of A. japonicus but poorly internalized into the coelomocyte under the tested conditions. It was thus postulated that V. splendidus was pathogenic to A. japonicus, mainly due to its extracellular products. The main extracellular proteins of V. splendidus were detected using MALDI-TOF-TOF/MS, and a metalloprotease was identified. The gene encoding a metalloprotease belonging to the thermolysin family, named vsm, was cloned and characterized. Furthermore, the expression of vsm was growth dependent and the supernatant of V. splendidus also showed high protease activity. The vsm gene was conditionally expressed in Escherichia coli BL21(DE3). Enzyme activity analysis showed that the optimal temperature and pH for purified recombinant Vsm were approximately 40 °C and 7.0, respectively. Furthermore, Vsm was determined to be associated with the pathogenesis of V. splendidus due to the following aspects: (1) real-time reverse transcriptase PCR showed that expression of vsm was significantly up-regulated when V. splendidus was co-incubated with the coelomic fluid of A. japonicus; and (2) purified recombinant Vsm showed obvious cytotoxicity to the coelomocyte of A. japonicus. Our results indicated that Vsm is involved in the interaction between V. splendidus and A. japonicus and also contributed to the cytotoxic effects on the coelomocyte of A. japonicus.

Cloning and characterization of Vshppd, a gene inducing haemolysis and immune response of Apostichopus japonicus

Vibrio splendidus is one of the most important opportunistic pathogens that are ubiquitously presented in the marine environment. It is the main pathogen of “skin ulcer syndrome” which occurs in Apostichopus japonicus; however, little is known about its virulence factors and pathogenic mechanism. In this study, V. splendidus isolated from diseased A. japonicus was determined to possess α-haemolytic activity, and a gene named Vshppd, encoding a 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) homologue from pathogenic V. splendidus, was cloned, and conditionally expressed in Escherichia coli. Haemolytic activity analyses of both purified protein and recombinant BL21(DE3)/pET258h revealed that Vshppd was responsible for the haemolysis of V. splendidus. What's more, expressions of Vshppd under various conditions were also determined using real-time reverse transcriptase PCR. Transcription of Vshppd was up-regulated by both iron chelator and coelomic fluid of A. japonicus, while it was down-regulated by glucose. Furthermore, DH5α/pBATh, an E. coli strain displaying Vshppd on the cell surface was genetically constructed and showed significantly stimulatory effects on the expression of immune-related genes encoding LBP and caspase of A. japonicus. Our results indicated that haemolysin Vshppd was not only involved in the interaction between V. splendidus and A. japonicus but also contributed to the stimulatory effect on immune response. Statement of relevanceV. splendidus is an important opportunistic pathogen that can infect a broad range of aquatic animals including A. japonicus, but little was known about its pathogenic mechanism. Our study firstly showed that Vshppd conferred the haemolytic activity of V. splendidus, involved in the interaction between V. splendidus and host, and contributed to the stimulatory effect on immune response.

RNA sequencing analysis to capture the transcriptome landscape during skin ulceration syndrome progression in sea cucumber Apostichopus japonicus.

BackgroundSea cucumber Apostichopus japonicus is an important economic species in China, which is affected by various diseases; skin ulceration syndrome (SUS) is the most serious. In this study, we characterized the transcriptomes in A. japonicus challenged with Vibrio splendidus to elucidate the changes in gene expression throughout the three stages of SUS progression.ResultsRNA sequencing of 21 cDNA libraries from various tissues and developmental stages of SUS-affected A. japonicus yielded 553 million raw reads, of which 542 million high-quality reads were generated by deep-sequencing using the Illumina HiSeq™ 2000 platform. The reference transcriptome comprised a combination of the Illumina reads, 454 sequencing data and Sanger sequences obtained from the public database to generate 93,163 unigenes (average length, 1,052 bp; N50 = 1,575 bp); 33,860 were annotated. Transcriptome comparisons between healthy and SUS-affected A. japonicus revealed greater differences in gene expression profiles in the body walls (BW) than in the intestines (Int), respiratory trees (RT) and coelomocytes (C). Clustering of expression models revealed stable up-regulation as the main pattern occurring in the BW throughout the three stages of SUS progression. Significantly affected pathways were associated with signal transduction, immune system, cellular processes, development and metabolism. Ninety-two differentially expressed genes (DEGs) were divided into four functional categories: attachment/pathogen recognition (17), inflammatory reactions (38), oxidative stress response (7) and apoptosis (30). Using quantitative real-time PCR, twenty representative DEGs were selected to validate the sequencing results. The Pearson’s correlation coefficient (R) of the 20 DEGs ranged from 0.811 to 0.999, which confirmed the consistency and accuracy between these two approaches.ConclusionsDynamic changes in global gene expression occur during SUS progression in A. japonicus. Elucidation of these changes is important in clarifying the molecular mechanisms associated with the development of SUS in sea cucumber.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2810-3) contains supplementary material, which is available to authorized users.

Characterization of Functional furVs as a Biomarker for Detection of Aquatic Pathogen Vibrio splendidus

AbstractVibrio splendidus is an important opportunistic pathogen that can infect a broad range of aquatic animals including Apostichopus japonicus, leading to skin ulceration syndrome. In this study, fur gene of V. splendidus (furVs), the gene encoding ferric uptake regulator (Fur) of V. splendidus, was cloned and characterized. furVs possessed the signature ammonia acids of Vibrio sp., and its mRNA level was regulated by iron and coelomic fluid of A. japonicus. While the amino acid sequence of the furVs showed high homology with the compared Fur proteins of other Vibrio sp., the nucleotide sequence of furVs differed greatly from that of the other fur genes from Vibrio sp., based on which a pair of specific primers VSFurRTF3 and VSFurRTR3, was designed. A distinct DNA band with a length of 223 bp can be amplified from V. splendidus with great specificity. The detection limits for V. splendidus in seawater, homogenized tissues of epidermis, muscles, and coelomic fluid from A. japonicus, was 1 × 102, 1 × 105, 1 × 103, and 1 × 102 CFU/mL, respectively. Moreover, the detection limit was as low as 8 × 10−3 pg/μL when using DNA of V. splendidus as a template. Our results provide a simple and rapid polymerase chain reaction method for the sensitive and specific detection of V. splendidus.

Identification and characterization of miR-31 potential targets by RNA-seq.

In our previous work, miR-31 displayed differential significant expression in Apostichopus japonicus sea cucumber with skin ulcer syndrome and modulated coelomocytes ROS production by targeting p105. To identify other promising targets ofmiR-31, 4 transcriptome libraries of coelomocytes, as well as 2 control libraries, were constructed frommiR-31 mimics (31M) or AMO-miR-31 (31I) and injected into a sea cucumber at 12 and 24h. A total of207,977 unigenes with an average length of 363bp were assembled, in which17,204 distinct sequences (8.27% of the unigenes) were successfully matched with annotated protein sequences. Fragments per kilobase of transcript per million fragments mapped analysis indicated that 1325 unigenes displayed up-regulated expression profiles in the 31I-12 group and were depressed in the 31M-12 group compared with the control group. A total of 1470 unigenes showed down-regulated expressions in 31I-12 and were induced in 31M-12. Similarly, 2079 and 2098 unigenes were detected at 24h post-injection. Among these unigenes, 36 unigenes (depressed expression in the 31M group and induced in the 31I group) showed consistent expression patterns at 2 examined time points and were considered promising targets of miR-31. qPCR analysis confirmed that all 4 unigenes showed opposite expression profiles to miR-31 in cultured coelomocytes. Our present work provided a fast and feasible method of identifying miR-31 targets by transcriptome analysis. The results of this study would enhance our present understanding ofmiR-31 function insea cucumber immune regulation.

Expression analysis of microRNAs related to the skin ulceration syndrome of sea cucumber Apostichopus japonicus.

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in many biological processes. To investigate the miRNAs related to skin ulceration syndrome (SUS) of Apostichopus japonicus, small RNA libraries of body wall, intestine, respiratory tree and coelomocytes from healthy and diseased A.japonicus were sequenced on Illumina Hiseq 2000 platform. A total of 247 conserved and 10 novel miRNAs were identified across all libraries. After pair-wise comparisons, 215 miRNAs in body wall, 36 in intestine, 2 in respiratory tree and 38 in coelomocytes showed significant expression differences. Further analyses were conducted on some tissue-specific differentially expressed miRNAs: miR-8 and miR-486-5p in body wall, miR-200-3p, let-7-5p and miR-125 in intestine, miR-278a-3p and bantam in respiratory, miR-10a and miR-184 in coelomocytes. Notably, these miRNAs in some species were reported to function in various physiological or pathological processes associated with immune regulations. Using stem-loop quantitative real time PCR, six representative miRNAs in four tissues were selected to validate the sequencing results. The Pearson's correlation coefficient (R) of the six miRNAs ranged from 0.777 to 0.948, which confirmed the consistency and accuracy between these two approaches. This study provides comprehensive expression and regulation patterns of functional miRNAs in different tissues and gives insights into the tissue-specific immune response mechanisms in SUS-infected A.japonicus.

Protective effects of chicken egg yolk antibody (IgY) against experimental Vibrio splendidus infection in the sea cucumber (Apostichopus japonicus).

Vibrio splendidus is one of the most harmful pathogens associated with skin ulceration syndrome in the sea cucumber (Apostichopus japonicus) due to its high virulence and frequency of appearance. The objective of this study was to determine the effectiveness of chicken egg yolk antibody (IgY) against V.splendidus infection in the sea cucumber. Whole V.splendidus cells were used as an immunogen to immunize 20 White Leghorn hens (25 weeks old). IgY was produced from egg yolks obtained from these immunized hens using water dilution, two-step salt precipitation and ultrafiltration. The purity of the IgY produced was approximately 83%. Enzyme-linked Immunosorbent Assay indicated a high specificity for IgY with a maximum antibody titer of 320,000. The growth of V.splendidus in liquid medium was significantly inhibited by IgY in a dose-dependent manner at concentrations ranging from 1 to 10mg/mL. The protective effects of IgY were evaluated in sea cucumber by intraperitoneally injecting anti-V.splendidus IgY antibodies (10mg/mL) or immersing the sea cucumber in aqueous IgY (1g/L) after an intraperitoneal injection of V.splendidus. Intraperitoneal injection resulted in an 80% survival while immersion resulted in a 75% survival during the 11-day experimental period. The survival rates were significantly higher than the positive control and the non-specific IgY group (P<0.05). As well, the bacterial burden in the respiratory tree, intestine and coelomic liquid was significantly (P<0.05) lower in sea cucumber treated with specific IgY than those treated with non-specific IgY. The phagocytosis of coelomocytes for V.splendidus in the presence of specific IgY was significantly (P<0.05) higher than that obtained with non-specific IgY or without IgY, suggesting that specific IgY enhanced phagocytic activity. The current work suggests that specific IgY has potential for protecting sea cucumbers against V.splendidus infection.

Efficiency of a bacteriophage in controlling vibrio infection in the juvenile sea cucumber Apostichopus japonicus

Vibro cyclitrophicus is thought to be responsible for the cause of severe infection in juvenile sea cucumber (Apostichopus japonicus). Increases in the prevalence of antibiotic resistant pathogens have promoted us to develop effective agents to antibiotics for combating microbial infection among animals as well as human. In the present study, we isolated a bacteriophage with the ability to cleave V. cyclitrophicus present in the sewage of sea cucumber farms. This bacteriophage was designated as phage vB_VcyS_Vc1 (Vibrio phage Vc1). A one-step growth curve analysis of the phage revealed eclipse and latent periods of 25 and 45min, respectively, with a burst size of 215PFU/infected cell. Morphological analysis revealed the phage belongs to family of Siphoviridae. Furthermore, genomic sequencing results revealed a double-stranded DNA containing 44,541bp with a G+C content of 44.16%. Forty four coding sequences were annotated in the genome, and nineteen of these were associated with a known function. Genes related to virulence and toxins were not detected in the genome. In addition, a prevention experiment conducted in a marine environment demonstrated that the phage increased the survival rate of juvenile sea cucumbers (18±2g) from 18% to 81% when the sea cucumber were fed with feedstuff containing the freeze-dried phage powder, 58% when the sea cucumber was injected with purified phages (MOI=10), and 63% when the sea cucumbers were immersed in a suspension containing purified phages. Notably, when the phage provided nearly the same protection to the sea cucumbers as antibiotic when it was fed to the sea cucumbers in the form of freeze-dried powder mixed with feedstuff. Taken together, the results demonstrated that the use of phage to control the infection of V. cyclitrophicus in sea cucumber may be a feasible alternative to antibiotics. Statement of relevanceThe A. japonicus (sea cucumber) is the most profitable aquaculture animal with the highest output as single variety in China, which is of high nutritional value and economic value. In the past decade, as the growth of the market demand, A. japonicus's artificial breeding scale expands rapidly and the sea cucumber industry has become a vigorous sector in China aquaculture. However, with the high density and intensive development model of A. japonicus breeding, the various diseases have resulted in serious economic losses. Skin ulceration syndrome (SUS) is one of the most epidemic and serious diseases that affect sea cucumbers. At present, sanitizer and antibiotics are still commonly used in farms or hatcheries to control the bacterial disease in juvenile sea cucumbers, which resulted in the environment pollution and antibiotic residues. Nowadays, increased appearance of antibiotic resistant phenomenon promoted us to develop effective agents. Hence, alternative strategies to antibiotics should be further developed. This study was the first to monitor the effectiveness of using feedstuff mixed with freeze-dried phage powder as a form of protection for sea cucumber against vibrio infection, and demonstrated the effectiveness of phage in the control of pathogen in aquaculture.

Transcriptome Analysis and Discovery of Genes Involved in Immune Pathways from Coelomocytes of Sea Cucumber (Apostichopus japonicus) after Vibrio splendidus Challenge.

Vibrio splendidus is identified as one of the major pathogenic factors for the skin ulceration syndrome in sea cucumber (Apostichopus japonicus), which has vastly limited the development of the sea cucumber culture industry. In order to screen the immune genes involving Vibrio splendidus challenge in sea cucumber and explore the molecular mechanism of this process, the related transcriptome and gene expression profiling of resistant and susceptible biotypes of sea cucumber with Vibrio splendidus challenge were collected for analysis. A total of 319,455,942 trimmed reads were obtained, which were assembled into 186,658 contigs. After that, 89,891 representative contigs (without isoform) were clustered. The analysis of the gene expression profiling identified 358 differentially expression genes (DEGs) in the bacterial-resistant group, and 102 DEGs in the bacterial-susceptible group, compared with that in control group. According to the reported references and annotation information from BLAST, GO and KEGG, 30 putative bacterial-resistant genes and 19 putative bacterial-susceptible genes were identified from DEGs. The qRT-PCR results were consistent with the RNA-Seq results. Furthermore, many DGEs were involved in immune signaling related pathways, such as Endocytosis, Lysosome, MAPK, Chemokine and the ERBB signaling pathway.

Management of Intravenous Contrast Extravasations with Ultrasonography: A Case Report

Extravasation of ionic and nonionic contrast materials is a well-recognized complication of contrast-enhanced imaging studies. Complications vary from minimal swelling to severe skin and subcutaneous ulceration, necrosis, and compartment syndrome. We report a case of Omnipaque (iohexol) extravasation in a 50-year-old man with erythema, blistering, and compartment syndrome who was treated medically but was not cured. Using gray scale ultrasonography, we determined the characteristics of the lesion, its distance from the skin, and its proximity to the vessels. We then determined the depth of the lesion, and then inserted the tip of the needle into the lesion. We also used ultrasonography in locations where extravasation was near an artery. After aspiration, the diameter of the lesion decreased significantly. The patient was cured by ultrasonography-guided aspiration from the extravasated site.

ITRAQ-based proteomics reveals novel members involved in pathogen challenge in sea cucumber Apostichopus japonicus.

Skin ulceration syndrome (SUS) is considered to be a major constraint for the stable development of Apostichopus japonicus culture industries. In this study, we investigated protein changes in the coelomocytes of A. japonicus challenged by Vibrio splendidus using isobaric tags for relative and absolute quantification (iTRAQ) over a 96 h time course. Consequently, 228 differentially expressed proteins were identified in two iTRAQs. A comparison of the protein expression profiles among different time points detected 125 proteins primarily involved in response to endogenous stimuli at 24 h. At 48 h, the number of differentially expressed proteins decreased to 67, with their primary function being oxidation reduction. At the end of pathogen infection, proteins responsive to amino acid stimuli and some metabolic processes were classified as the predominant group. Fifteen proteins were differentially expressed at all time points, among which eight proteins related to pathologies in higher animals were shown to be down-regulated after V. splendidus infection: paxillin, fascin-2, aggrecan, ololfactomedin-1, nesprin-3, a disintegrin-like and metallopeptidase with thrombospondin type 1 motif (Adamts7), C-type lectin domain family 4 (Clec4g) and n-myc downstream regulated gene 1 (Ndrg1). To gain more insight into two SUS-related miRNA (miR-31 and miR-2008) targets at the protein level, all 129 down-regulated proteins were further analyzed in combination with RNA-seq. Twelve and eight proteins were identified as putative targets for miR-31 and miR-2008, respectively, in which six proteins (5 for miR-31 and 1 for miR-2008) displayed higher possibilities to be regulated at the level of translation. Overall, the present work enhances our understanding of the process of V. splendidus-challenged sea cucumber and provides a new method for screening miRNAs targets at the translation level.

Proteomic identification of differentially expressed proteins in sea cucumber Apostichopus japonicus coelomocytes after Vibrio splendidus infection

Skin ulceration syndrome (SUS) was the main limitation in the development of Apostichopus japonicus culture industries. To better understand how Vibrio splendidus modulates SUS outbreak, the immune response of A. japonicus coelomocytes after the pathogen challenge were investigated through comparative proteomics approach, and differentially expressed proteins were screened and characterized in the present study. A total of 40 protein spots representing 30 entries were identified at 24, 72 and 96h post-infection. Of these proteins, 32 were up-regulated and 8 were down-regulated in the V. splendidus challenged samples compared to those of control. These differentially expressed proteins were mainly classified into four categories by GO analysis, in which approximate 33% of proteins showed to be related to immunity response. The mRNA expression levels of 6 differentially expressed proteins were further validated by qRT-PCR. Similar protein-mRNA-level expression patterns were detected in genes of phospholipase (spot 4), G protein (spot 20), annexin (spot 30) and filamin (spot 31). Whilst the levels of ficolin (spot 12) and calumenin (spot 14) transcripts were not corresponded with those of their translation products. These data provide a new insight to understand the molecular immune mechanism of sea cucumber responsive towards pathogen infection.

Divergent Metabolic Responses of Apostichopus japonicus Suffered from Skin Ulceration Syndrome and Pathogen Challenge

Skin ulceration syndrome (SUS) is the main limitation in the development of Apostichopus japonicus culture industries, in which Vibrio splendidus has been well documented as one of the major pathogens. However, the intrinsic mechanisms toward pathogen challenge and disease outbreak remain largely unknown at the metabolic level. In this work, the metabolic responses were investigated in muscles of sea cucumber among natural SUS-diseased and V. splendidus-challenged samples. The pathogen did not induce obvious biological effects in A. japonicus samples after infection for the first 24 h. An enhanced energy storage (or reduced energy demand) and immune responses were observed in V. splendidus-challenged A. japonicus samples at 48 h, as marked by increased glucose and branched chain amino acids, respectively. Afterward, infection of V. splendidus induced significant increases in energy demand in A. japonicus samples at both 72 and 96 h, confirmed by decreased glucose and glycogen, and increased ATP. Surprisingly, high levels of glycogen and glucose and low levels of threonine, alanine, arginine, glutamate, glutamine, taurine and ATP were founded in natural SUS-diseased sea cucumber. Our present results provided essential metabolic information about host-pathogen interaction for sea cucumber, and informed that the metabolic biomarkers induced by V. splendidus were not usable for the prediction of SUS disease in practice.

De Novo Assembly of the Sea Cucumber Apostichopus japonicus Hemocytes Transcriptome to Identify miRNA Targets Associated with Skin Ulceration Syndrome

Background De novo transcriptome sequencing is a robust method of predicting miRNA target genes, especially samples without reference genomes. Differentially expressed miRNAs have been previously identified in hemocytes collected from healthy skin and from skin affected by skin ulceration syndrome (SUS) in Apostichopus japonicus . Target identification for these differentially expressed miRNAs is a major challenge for this non-model organism.Methodology/Principal FindingsTo thoroughly understand the function of miRNAs, a normalized cDNA library was sequenced with the Illumina Hiseq2000 technology. A total of 91,098,474 clean reads corresponding to 251,148 unigenes, each with an average length of 494bp, were obtained. Blastx analysis against a nonredundant (nr) NCBI protein database revealed that in this set, 52,680 unigenes coded for 3,893 annotated proteins. Two digital gene expression (DGE) libraries from healthy and SUS samples showed that 4,858 of the unigenes were expressed at significantly different levels; 2,163 were significantly up-regulated, while 2,695 were significantly down-regulated. The computational prediction of miRNA targets from these differentially expressed genes identified 732 unigenes as the targets of 57 conserved and 8 putative novel miRNA families, including spu-miRNA-31 and spu-miRNA-2008.ConclusionThis study demonstrates the feasibility of identifying miRNA targets by transcriptome analysis. The DGE assembly data represent a substantial increase in the genomic resources available for this species and will provide insights into the gene expression profile analysis and the miRNAs function annotations of further studies.

Extravasation of contrast media managed with recombinant human hyaluronidase

In minor cases, contrast extravasation may cause pain, swelling, and localized erythema. However, in more severe cases, extensive tissue and skin necrosis, ulceration, and compartment syndrome may occur, often necessitating a surgical consultation. Hyaluronidase has been used successfully in the management of extravasated contrast media in several reports. In addition, recombinant human hyaluronidase is approved for use as an adjunct in subcutaneous urography for improving resorption of radiopaque agents. In this case, a 57-year-old white female admitted for a chronic obstructive pulmonary disease exacerbation with pneumonitis and hypoxic respiratory failure experienced contrast extravasation during a computed tomographic scan of her chest. Approximately 100 mL of iodinated contrast extravasated into the right antecubital fossa, infiltrating approximately the distal two-thirds of the upper arm. Five 150-U vials of recombinant human hyaluronidase were injected in 150-U aliquots in a circle around the extravasation site using a 27-gauge needle 1 hour after the extravasation occurred. In a follow-up 4 hours later, marked improvement was observed. Sixteen hours after hyaluronidase treatment, the tissue had reverted to a near-normal state with no pain, erythema, swelling, or tenderness noted. Hyaluronidase successfully treated this extravasation of a large volume of iodinated contrast and appears to be a reasonable treatment option for more extensive subcutaneous contrast media extravasations.

Characterization of skin ulceration syndrome associated microRNAs in sea cucumber Apostichopus japonicus by deep sequencing

MicroRNAs (miRNAs) constitute a family of small RNA species which have been demonstrated to be one of key effectors in mediating host–pathogen interaction. In this study, two haemocytes miRNA libraries were constructed with deep sequenced by illumina Hiseq2000 from healthy (L1) and skin ulceration syndrome Apostichopus japonicus (L2). The high throughput solexa sequencing resulted in 9,579,038 and 7,742,558 clean data from L1 and L2, respectively. Sequences analysis revealed that 40 conserved miRNAs were found in both libraries, in which let-7 and mir-125 were speculated to be clustered together and expressed accordingly. Eighty-six miRNA candidates were also identified by reference genome search and stem-loop structure prediction. Importantly, mir-31 and mir-2008 displayed significant differential expression between the two libraries according to FPKM model, which might be considered as promising targets for elucidating the intrinsic mechanism of skin ulceration syndrome outbreak in the species.

Isolation and pathogenicity of pathogens from skin ulceration disease and viscera ejection syndrome of the sea cucumber Apostichopus japonicus

The diseases of cultured sea cucumber have been the bottle-neck for the sustainable development of sea cucumber ( Apostichopus japonicus) cultivation in China. Two diseases that occurred from 2004 and caused mass mortalities of sea cucumbers are still prevalent in Liaoning and Shandong provinces in China. One is skin ulceration syndrome and the other is viscera rejection syndrome. The epidemiology and pathogenicity of the diseases were investigated. Microscopic observations were made on healthy and diseased individuals of sea cucumbers to record the bacteria, mold and parasites on the surface of body wall, and the relationship of microbes between skin ulceration disease and viscera ejection disease. The two diseases expressed several differences. Six predominant isolates of bacteria (2004A, 2004B, 04101, 04102, 04103 and 04104) were isolated from the sea cucumbers suffering from skin ulceration in indoor ponds for wintering and outdoor ponds for culturing. Two isolates of bacteria (041201 and 041202) were isolated from the sea cucumbers suffering from viscera ejection syndrome in indoor wintering ponds. Induced infection experiments with these isolates of bacteria showed that all eight predominant isolates of bacteria were pathogenic as they showed the same signs as naturally infected and re-isolates from diseased sea cucumber had the same morphological characteristics. Physiological and biochemical tests and the sequence similarity analysis of the 16S rRNA genes of these bacterial isolates indicated that isolate 2004A was similar to Vibrio cyclitrophicus, 2004B similar to V. splendidus, isolates 04101 and 04103 similar to V. harveyi, 04102 similar to V. tasmaniensis, isolate 04104 similar to Photobacterium sp. isolate 041201 similar to Arthrobacter protophormiae, and isolate 041202 similar to Staphylococcus equorum. Antibiotic sensitive test showed that the eight isolates of bacteria were sensitive to Ofloxacin, Levofloxacin hydrochloride, Cefobid, Doxycycline, Novobiocin, but not sensitive to Oxacillin sodium, Aminobenzylpenicillin, Spectinomycin, or Cefradine.

Isolation and pathogenicity of pathogens from skin ulceration and viscera ejection syndrome of sea cucumber

Isolation and pathogenicity of pathogens from skin ulceration and viscera ejection syndrome of sea cucumber

Compartment syndrome in the hand due to extravasation of contrast material

Extravasation of contrast material is a well-recognized complication of contrast-enhanced imaging studies. Most extravasations result only in minimal swelling or erythema; however, severe skin necrosis, ulceration and compartment syndrome may occur with extravasation of large volumes. This article presents a patient in whom extravasation developed after computed tomography (CT) contrast material was injected intravenously in the dorsum of the hand. Compartment syndrome was diagnosed, and the patient underwent fasciotomy. In follow-up, the patient regained full use of the hand. Although the use of contrast material has significantly improved the diagnostic accuracy of CT, anaphylaxis and contrast material extravasation are important complications. Selection of non-ionic contrast material, careful evaluation of the intravenous administration site and close monitoring of the patient during contrast material injection may help minimize or prevent extravasation injuries.