Development of a 38 K single nucleotide polymorphism array and application in genomic selection for resistance against Vibrio harveyi in Chinese tongue sole,Cynoglossus semilaevis

Based on 1572 re-sequenced Chinese tongue sole (Cynoglossus semilaevis), we investigated the accuracy of four genomic methods at predicting genomic estimated breeding values (GEBVs) of Vibrio harveyi resistance in C. semilaevis when SNPs varying from 500 to 500 k. All methods outperformed the pedigree-based best linear unbiased prediction when SNPs reached 50 k or more. Then, we developed an SNP array “Solechip No.1” for C. semilaevis breeding using the Affymetrix Axiom technology. This array contains 38,295 SNPs with an average of 10.5 kb inter-spacing between two adjacent SNPs. We selected 44 candidates as the parents of 23 families and genotyped them by the array. The challenge survival rates of offspring families had a correlation of 0.706 with the mid-parental GEBVs. This SNP array is a convenient and reliable tool in genotyping, which could be used for improving V. harveyi resistance in C. semilaevis coupled with the genomic selection methods.     

Differential resistance to edwardsiellosis in rohu (<Emphasis Type="Italic">Labeo rohita</Emphasis>) families selected previously for higher growth and/or aeromoniasis-resistance

Selection in fish for disease resistance is one of the most useful approaches to solve disease problems. Genetic variation in resistance to edwardsiellosis in fullsib families of rohu, Labeo rohita was investigated in the present study. A large variation in the susceptibility pattern (0 to 94.74 percent survival) against Edwardsiella tarda challenge was observed among 57 families. Additive genetic variation showed a heritability of 0.38 ± 0.08 across the year-class survival. The apparent resistant families showed more delayed mortality than the apparent susceptible ones. The cross-protection provided by aeromoniasis-resistant lines of rohu to edwardsiellosis was also studied to evaluate the possibility of selection for both diseases simultaneously. Challenge of F1-generation aeromoniasis-resistant and -susceptible lines with E. tarda showed significant difference in survival between the lines with higher percent survival in resistant line. This study suggests that direct selection method may be used reliably in selection programs and selection for multiple diseases simultaneously can be considered for rohu.     

研究报告: 基于核酸适配体的胶体金比色法检测鳗弧菌

目的 鳗弧菌（Vibrio anguillarum）可引起鲑鱼、鳗鲡、鲈鱼和牙鲆等多种水产养殖动物的疾病，是水产养殖中的一种重要病原菌，对其进行快速检测是确保水产养殖安全和食品安全所必需的。方法 本文利用鳗弧菌与其核酸适配体之间较强的亲和力，通过鳗弧菌夺取胶体金颗粒表面的核酸适配体，使胶体金溶液的吸光度发生变化，从而建立一种可定量检测鳗弧菌的方法。结果 该方法对鳗弧菌的吸光度值显著高于对溶藻弧菌、铜绿假单胞菌、变形假单胞菌、嗜水气单胞菌和迟钝爱德华氏菌等非目标菌的吸光度值（P<0.01），并在1~10⁵ CFU/ml的检测范围内呈现较好的线性关系。用该方法对不同盐度和鱼体组织样品进行加标回收检测，结果显示回收率和相对标准偏差等指标都符合相应检测标准。结论 该检测方法对鳗弧菌有较好的特异性，可用于水产品或食品中鳗弧菌的定量检测。     

Phenotypic diversity of Edwardsiella tarda isolated from different origins

Aims: To evaluate the diversity of phenotypic characteristics among isolates of Edwardsiella tarda from various origins. Methods and results: A total of 10 E. tarda strains were investigated on biological characteristics including flagella formation, bacterial motility, biofilm formation, extracellular protein and plasmid profiles. All the E. tarda strains (including two previous recognized as nonflagellation strains) were proven to have an average of 1–7 peritrichous flagella with the precise number positively correlated with motility and biofilm formation. All the E. tarda strains exhibited similar protein profiles except ET2034, LMG2793 and ET080814, which lacked the three major bands of approximately 18, 21 and 55 kDa. E. tarda with the same geographic location shared similar plasmid profiles. Conclusions:  Edwardsiella tarda strains exhibited diversities in phenotypic characteristics that may be linked to differences in geographic location or host origin. In addition, the number of flagella is essential for bacterial motility and biofilm formation. Significance and Impact of the Study: This is the first report demonstrating the difference in flagella formation between E. tarda strains, which may broaden the understanding of flagellation trait at intra‐species level. Furthermore, evaluation of virulence‐associated characteristics can provide useful information for unveiling the diverse pathogenic mechanisms of E. tarda.     

Gene cloning and expression analysis of IRF1 in half-smooth tongue sole (Cynoglossus semilaevis)

Interferon regulatory factor 1 (IRF1) was known to play key roles in antiviral defense in several species, and some other important biological processes. In this report, full length cDNA of IRF1 from Cynoglossus semilaevis (CsIRF1) was identified. It was of 1,455 bp, containing a 5′ UTR of 104 bp, a 3′ UTR of 541 bp with a poly (A) tail and an ORF of 810 bp encoding a putative protein of 269 amino acids. The putative CsIRF1protein contained one conserved IRF domain (1–113aa), and two low complexity regions (140–158aa and 230–242aa, respectively). Phylogenetic analysis showed that CsIRF1 was conserved in the teleost evolutionary branch, which was independent of mammalian, birds and amphibians. Additionally, CsIRF1 had the 96 % homology with marine fishes, while 66 % with freshwater fishes. The expression profiles of CsIRF1was analyzed by quantitative real-time PCR in healthy tissues and in immune tissues challenged with different pathogens [Vibrio anguillarum and Lymphocystis disease virus (LCDV)], respectively. CsIRF1 was widely expressed in healthy tissues of Cynoglossus semilaevis and with the highest expression in blood, as much as 19 times of that in liver. V. anguillarum and LCDV both induced the CsIRF1 gene expression distinctly in liver, with the peak value reached to 98-fold at 6 h and 25-fold at 24 h, respectively. The bacteria induced CsIRF1 suddenly up-expression in each detected tissues. However, at the initial stage of the challenge of virus LCDV, the CsIRF1 expression in blood and spleen were up regulated; on the contrary, its expression in liver and head kidney were down regulated, 0.3 and 0.4-fold 6 h post virus injection, respectively. These results suggested that CsIRF1 gene might involve in not only antiviral activity but also antibacterial procedure, indicating its vital role in Cynoglossus semilaevis innate defense system.     

Secreted glyceraldehyde-3-phosphate dehydrogenase as a broad spectrum vaccine candidate against microbial infection in aquaculture

Aims: Subcellullar localizations and cross‐immunities of GAPDHs from six common pathogenic bacteria in aquaculture were investigated. Methods and Results: Subcellullar localizations of GAPDHs of Edwardsiella tarda EIB202, Edwardsiella ictaluri ATCC33202, Aeromonas hydrophila LSA34, Vibrio anguillarum MVM425, Vibrio alginolyticus EPGS020401 and Vibrio harveyi VIB647 were analysed with Western blotting, indirect immunofluorescence and flow cytometry examinations. Immunoprotections of different recombinant GAPDHs against these pathogens were investigated with zebrafish model. Western blotting of subcellular extractions showed that all GAPDHs were secreted into extracellular medium and periplasmic space. In addition, GAPDHs were demonstrated to distribute in the outer membranes except MVM425 and VIB647. And, GAPDHs were confirmed to be present on the surface of these bacteria with indirect immunofluorescence and flow cytometry examinations. The remarkable cross‐protective immunities of these recombinant GAPDHs were induced in zebrafish, and the relative protective survivals were almost over 60%. Conclusions: Localizations of GAPDHs from these pathogenic bacteria were similar to many other causative agents. And, GAPDHs could be important protective antigens and give remarkable cross‐immunity against different pathogens. Significance and Impact of the Study: Recombinant GAPDH could be designed as a broad spectrum vaccine candidate against multiple microbial infections in aquaculture.     

Study on the immune enhancement of different immunoadjuvants used in the pentavalent vaccine for turbots

In this study, we investigated the immune enhancing effects of different adjuvants used in a pentavalent vaccine for turbots. The pentavalent vaccine consisted of inactive bacterial cells from five common pathogenic strains (Vibrio anguillarum, Vibrio scophtalmi, Edwardsiella tarda, Vibrio harveyi and Vibrio alginolyticus) and the adjuvants were astragalus polysaccharides (APS), propolis, and the Freund’s complete adjuvant (FCA). Turbots were immunized with the pentavalent vaccine alone or with one of the adjuvants, and the immune efficiency was evaluated by measuring the activities of lysozyme (LSZ) and superoxide dismutase (SOD), and serum antibody titers. Fish were also challenged with the pathogens after immunization and the relative percent survival (RPS) was assessed. Our results showed that APS, propolis, and FCA had significant immune-enhancing effects on turbots as shown by the higher titers of antibodies against the pathogens, increased LSZ and SOD activities, and enhanced RPS after challenge with pathogens. Among the three adjuvants, FCA had the most significant immune synergistic effects with the vaccine, and APS and propolis had lower and similar immune synergies.     

Genetic studies to re-affiliate Edwardsiella tarda fish isolates to Edwardsiella piscicida and Edwardsiella anguillarum species

Until 2012, the genus Edwardsiella was composed by three species Edwardsiella tarda, Edwardsiella hoshinae and Edwardsiella ictaluri. In 2013, Edwardsiella piscicida, compiling fish pathogenic strains previously identified as E. tarda was described, and more recently a new species isolated from diseased eel was reported, namely Edwardsiella anguillarum.The incorporation of these species into the genus makes necessary a revision of the taxonomic position of the isolates previously identified as E. tarda. Using AFLP technique, MLSA studies and in silico DNA–DNA hybridization, 46 of 49 E. tarda isolates were re-assigned as E. piscicida and 2 as E. anguillarum, whereas it was confirmed previous classification of the Edwardsiella types and reference strains used. The study of the taxonomic resolution of the genes 16S rRNA, adk, atpD, dnaJ, glnA, hsp60, tuf as well as the possible combinations among housekeeping genes, showed that the gene dnaJ was the more resolutive. In conclusion, the use of molecular techniques is necessary to accurately identify Edwardsiella isolates, especially when differentiating new species from E. tarda.     

Production of antibacterials from the freshwater alga Euglena viridis (Ehren)

The antibacterial properties of Euglena viridis, collected from a freshwater pond at the Central Institute of Freshwater Aquaculture (CIFA), Bhubaneshwar, India, were tested against various strains of virulent pathogens viz. Pseudomonas putida(PP1, PP2),P. aeruginosa (PA1, PA2, PA3, PA4), P. fluorescens (PF1, PF2, PF3, PF4), Aeromonas hydrophila (AH30, AH31, AH32, AH34), Edwardsiella tarda, Vibrio alginolyticus (VA1),V. anguillarum(VN1, VN2 & VN3), V. fluvialis (VF1), V. parahemolyticus (VP1) and V. harveyi (VH1) andEscherichia coli(O115, O1, O156, O164, O111 & O109). Four organic extracts viz. methanolic, ethanolic, acetone and acetone/ethanol of theE. viridis showed moderate to high antibacterial activity to all the bacterial pathogens. Rotavapor extraction products showed higher sensitivity in comparison to cold and hot extractions.     

Large scale SNP unearthing and genetic architecture analysis in sea-captured and cultured populations of Cynoglossus semilaevis

Knowledge on population structure and genetic diversity is a focal point for association mapping studies and genomic selection. Genotyping by sequencing (GBS) represents an innovative method for large scale SNP detection and genotyping of genetic resources. Here we used the GBS approach for the genome-wide identification of SNPs in a collection of Cynoglossus semilaevis and for the assessment of the level of genetic diversity in C. semilaevis genotypes. GBS analysis generated a total of 55.12 Gb high-quality sequence data, with an average of 0.63 Gb per sample. The total number of SNP markers was 563, 109. In order to explore the genetic diversity of C. semilaevis and to select a minimal core set representing most of the total genetic variation with minimum redundancy, C. semilaevis sequences were analyzed using high quality SNPs. Based on hierarchical clustering, it was possible to divide the collection into 2 clusters. The marine fishing populations were clustered and clearly separated from the cultured populations, and the cultured populations from Hebei was also distinct from the other two local populations. These analyses showed that genotypes were clustered based on species-related features. Differential significant SNPs were also captured and validated by GBS and SNaPshot, with linkage disequilibrium and haplotype analysis, seven SNPs have been confirmed to have obvious differentiation in two populations, which may be used as the characteristic evaluation sites of sea-captured and cultured Cynoglossus semilaevis populations. And SNP markers and information on population structure developed in this study will undoubtedly support genome-wide association mapping studies and marker-assisted selection programs. These differential SNPs could be also employed as the characteristic evaluation sites of sea-captured and cultured Cynoglossus semilaevis populations in future.     

Evaluation of the selective and differential ET medium for detection of Edwardsiella tarda in aquaculture systems

Aims: Edwardsiella tarda is an important pathogen in aquaculture where it can cause serious losses. A rapid detection of it is vital to minimize the mortalities caused by this disease, and in this work, the effectiveness of the selective differential Edw. tarda medium (ET) was evaluated for the diagnosis of edwardsiellosis as well as for its possible use in epidemiological studies. Methods and Results: ET medium was evaluated in parallel with the commercial Salmonella–Shigella agar (SS), which is usually employed for the selective isolation of enteric bacilli. Moreover, two general media (TSA‐1 and MA) were employed as a control. The results obtained showed that ET is distinctly selective for the isolation of Edw. tarda, allowing its recovery from mixed cultures and natural samples as a unique species. In contrast, although colonies of Edw. tarda could be clearly distinguishable in SS because of the appearance of a characteristic black centre, other enteric and nonenteric bacterial species were also able to grow on this medium. Conclusions: We recommend ET agar as an useful medium for the primary isolation of Edw. tarda from aquaculture samples. Significance and Impact of the Study: The results obtained support ET medium as the most appropriate to develop epidemiological studies of edwardsiellosis in aquaculture and permits an earlier diagnosis of this important disease.     

Prevalence of <Emphasis Type="Italic">Edwardsiella tarda</Emphasis> in Antarctic wildlife

For many years, the Antarctic region has been isolated from human activity. However, there is little data available regarding endemic and exotic diseases. The purpose of this work was to determine the prevalence of Edwardsiella tarda in Antarctic wildlife, including birds, mammals and fish. During the summer of 2000 and 2002 in the Potter Peninsula, and during the summer of 2001 and 2003 in Hope Bay, a total of 1,805 faecal samples from Antarctic animals and 50 infertile eggs of Adelie penguins (Pygoscelis adeliae) were collected in order to isolate E. tarda. The classic Edwardsiella tarda was isolated from 281 (15.1%) of the 1,855 Antarctic wildlife samples. This is the first report of E. tarda isolation from southern giant petrels (Macronectes giganteus), brown skuas (Stercorarius lonnbergi), south polar skuas (Stercorarius maccormicki), kelp gulls (Larus dominicanus), greater sheathbills (Chionis albus), chinstrap penguins (Pygoscelis antarctica), eggs of Adelie penguins and Weddell seals (Leptonychotes weddelli). None of the evaluated animals showed clinical signs of disease. Our results suggest that E. tarda is a common bacterium amongst Antarctic birds and mammals.     

Phenotypic traits,virulence‐associated gene profile and genetic relatedness of Edwardsiella tarda isolates from Japanese eel Anguilla japonica in Korea

Edwardsiella tarda is the predominant bacterium in farm‐cultured eel in Korea. Here, we evaluated the heterogeneity of 37 E. tarda isolates derived from Japanese eel with various origins (olive flounder, common carp and ornamental fish) between 2003 and 2010. Regardless of origins, the biochemical characteristics of E. tarda isolates were homogenous except hydrogen sulfide production, citrate utilization and mannitol fermentation. Based on the phylogenetic analysis of 16S rRNA, E. tarda isolates could be classified into two subgroups and displayed a close relation with Edwardsiella ictaluri and Edwardsiella hosinae lineages, suggesting that the subgroup I has been a predominant type in the Jeonnam and Jeonbuk provinces. I‐CeuI‐based pulsed‐field gel electrophoresis (PFGE) typing showed that the isolates from Japanese eels belonged to 11 pulsotypes, indicating that the presence of highly genomic diversity. Additionally, two isolates, ET‐060 and ET‐191, showed a high frequency of virulence genes (100%) and caused 90% and 60% mortality in Japanese eel, respectively. This finding suggests a substantial congruence of virulence gene profiles and pathogenicity. Our results demonstrate that the intraspecific diversity within E. tarda strains from Japanese eel has been in prior existence.

Significance and Impact of the Study

Based on the biochemical characteristics, the phylogenetic property of the 16S rRNA gene and PFGE types of Edwardsiella tarda, we could identify the intraspecific diversity of isolates from Japanese eel, Anguilla japonica in Korea. In addition, this study describes the strong congruence of virulence‐related genes and pathogenicity, suggesting that the virulence profile may be useful tool for prediction of pathogenicity.     

A first set of gene-derived polymorphic microsatellite markers in half-smooth tongue sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>)

Half-smooth tongue sole (Cynoglossus semilaevis) is a commercially important marine fish species in China. A set of type I microsatellite markers were identified through bioinformatic mining of the GenBank database. Thirteen of these markers showed polymorphisms through genotyping a sample of 30 individuals. A total of 47 alleles were detected, with an average of 3.6 alleles per locus. The number of alleles, observed and expected heterozygosity per locus ranged from two to five, from 0.14 to 0.93 and from 0.27 to 0.77, respectively. Three loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.0038) and no significant linkage disequilibrum between pairs of loci was found. The markers identified in this study will contribute to construction of genetic linkage maps and mapping of quantitative trait loci (QTL) of C. semilaevis.     

Genetic relationships of Edwardsiella strains isolated in China aquaculture revealed by rep-PCR genomic fingerprinting and investigation of Edwardsiella virulence genes

Aims: The aim of this study was to classify Edwardsiella strains isolated from China aquaculture based on biochemical and molecular methods. Methods and Results: In this study, biochemical characterization of 19 Edwardsiella tarda isolates and two Edwardsiella ictaluri isolates was performed with API 20E system. Other pathogenicity‐related phenotypes such as haemagglutination, haemolytic activities and lethality to fish were also examined in these strains. As it was difficult to categorize the subgroups of Edw. tarda according to their origins or phenotypic properties, three PCR‐based methods, i.e. PCR amplification of virulence genes, Enterobacterial repetitive intergenic consensus‐PCR and BOX‐PCR, were carried out to further resolve the relatedness of the Edw. tarda isolates. As a result, all Edw. tarda isolates could be generally grouped into pathogenic and nonpathogenic branches before being classified into strain‐specific or origin‐specific clades. Conclusions: Biochemical characterization was sensitive for interspecific typing, while PCR‐based approaches permitted a more accurate discrimination for intraspecific typing resulting in pathogenic and nonpathogenic clusters and further more delicate clades for Edwardsiella. Significance and Impact of the Study: PCR‐based genomic fingerprinting to study the relatedness and trace the pathogenicity of the Edwardsiella strains will be helpful in investigating the virulence factors of Edwardsiella and in the development of vaccines and diagnostics for edwardsiellosis.     

Overexpression of mltA in Edwardsiella tarda reduces resistance to antibiotics and enhances lethality in zebra fish

Aims: The aim of this study was to investigate the role of membrane‐bound lytic murein transglycosylase A (MltA) in a bacterial fish pathogen Edwardsiella tarda. Methods and Results: An mltA in‐frame deletion mutant (ΔmltA) and an mltA overexpression strain (mltA⁺) of Edw. tarda were constructed through double‐crossover allelic exchange and by transformation of a low‐copy plasmid carrying the intact mltA into the ΔmltA mutant, respectively. Either inactivation or overexpression of MltA in Edw. tarda resulted in elevated sensitivity to β‐lactam antibiotics and lower viability in oligotrophic or high osmotic environment than wild‐type strain. Autolysis induced by EDTA was reduced in ΔmltA strain, while mltA⁺ strain was virtually flimsy, indicating that MltA is responsible for the lysis effect. Moreover, mltA⁺ strain exhibited significant increases in lipopolysaccharide (LPS) biosynthesis and virulence to zebra fish compared with wild‐type strain. Conclusions: The results indicated that MltA plays essential roles in β‐lactam antibiotics and environmental stresses resistance, autolysis, LPS biosynthesis and pathogenicity of Edw. tarda. This is the first report that MltA has a virulence‐related function in Edw. tarda. Significance and Impact of the Study: This study provided useful information for further studies on pathogenesis of Edw. tarda.     

Detection of type III secretion gene as an indicator for pathogenic Edwardsiella tarda

Aims: To differentiate pathogenic and nonpathogenic Edwardsiella tarda strains based on the detection of type III secretion system (T3SS) gene using polymerase chain reaction (PCR). Methods and Results: Primers were designed to amplify Edw. tarda T3SS component gene esaV, catalase gene katB, haemolysin gene hlyA and 16S rRNA gene as an internal positive control. Genomic DNAs were extracted using a commercial isolation kit from 36 Edw. tarda strains consisting of 18 pathogenic and 18 nonpathogenic strains, and 50 ng of each DNA was used as the template for PCR amplification. PCR was performed with a thermocycler (TaKaRa TP600) in a 25‐μl volume. Products of esaV were detected in all pathogenic strains, but not in nonpathogenic strains; katB was detected in all pathogenic strains and one of nonpathogenic strains; hlyA was not detected in any strains. Conclusions: The detection of esaV gene can be used for the assessment of pathogenic Edw. tarda strains. Significance and Impact of the Study: The strategy using T3SS gene as the virulence indicator provides a useful tool for the clinical assessment of pathogenic Edw. tarda strains and prediction of edwardsiellosis risk in fish culture environments.     

Recombinant outer membrane protein A (OmpA) of Edwardsiella tarda, a potential vaccine candidate for fish, common carp

Outer membrane protein A (OmpA) is a component of the outer membrane of Edwardsiella tarda and is wildly distributed in Enterobacteriaceae family. The gene encoding the OmpA protein was cloned from E. tarda and expressed in Escherichia coli M15 cells. The recombinant OmpA protein containing His₆ residues was estimated to have a molecular weight of ∼38 kDa. In Western blot the native protein showed expression at ∼36 kDa molecular weight which was within the range of major outer membrane proteins (36–44 kDa) observed in this study. All E. tarda isolates tested harbored the ompA gene and the antibody raised to this protein was seen to cross react with other Gram negative bacteria. The OmpA protein characterized in this study was observed to be highly immunogenic in both rabbit and fish. In Enzyme linked immunosorbent assay, rabbit antisera showed an antibody titer of 1: 128,000. Common carp vaccinated with recombinant OmpA protein elicited high antibody production and immunized fish showed a relative percentage survival of 54.3 on challenge.