Sialic acid-binding lectins (SABLs) from Solen grandis function as PRRs ensuring immune recognition and bacterial clearance.

Sialic acid-binding lectins (SABLs) are ubiquitous ancient molecules with binding properties to N-acetyl or N-glycolyl carbohydrates, and play crucial roles in both adaptive and innate immune responses. In present study, recombinant protein and antibodies of two SABLs from mollusk Solen grandis (SgSABL-1 and SgSABL-2) were prepared to investigate their functions in innate immunity. The recombinant protein of SgSABL-1 (rSgSABL-1) could bind LPS, PGN and ß-glucan in vitro, while rSgSABL-2 could only bind PGN rather than LPS and ß-glucan. Be coincident with their PAMPs recognition properties, rSgSABL-1 displayed a broad agglutination spectrum towards gram-positive bacteria Micrococcus luteus, gram-negative bacteria Listonella anguillarum and fungi Pichia pastoris, and rSgSABL-2 only showed remarkable agglutinative effect on M. luteus and L. anguillarum. More importantly, after PAMPs recognition, rSgSABL-1 and rSgSABL-2 enhanced phagocytosis as well as encapsulation ability of hemocytes in vitro, and the enhanced encapsulation could be blocked by specific antibodies. All these results indicated that SgSABL-1 and SgSABL-2 functioned as two compensative pattern-recognition receptor (PRRs) with distinct recognition spectrum and involved in the innate immune response of S. grandis.

Polymorphism in a serine protease inhibitor gene and its association with the resistance of bay scallop (Argopecten irradians) to Listonella anguillarum challenge.

Serine protease inhibitors (SPIs) play a crucial role in regulation of both host and bacterial serine protease. They are classified into several protein families, where Kazal-type inhibitors are one of families with multi-domain. In the present study, the polymorphism of AiSPI from Bay scallop Argopecten irradians was found to be associated with disease resistance of bay scallop against Listonella anguillarum. Nine single nucleotide polymorphisms (SNPs) were identified in the exon region of AiSPI, where five SNPs were non-synonymous mutation. Three of these mutations were located in "kazal-like 3"domain, two SNP loci positioned at +536, +1312 were selected for further association studies. For the locus +536, the genotype frequency of A/G in the resistant stock (12.8%) was significantly lower (p < 0.05) than that in the susceptible stock (35.1%), while, the genotype A/A in the resistant stock (87.2%) was significantly higher in comparison with susceptible stock (64.9%) (p < 0.05). The G allele frequencies were 6.4% and 17.6% in resistant stock and susceptible stock, respectively, and χ2-test revealed a significant difference in the frequency distribution between the two stocks (p < 0.05). But there was no significant association between the mutation C-T at locus +1312 with either resistant or susceptible group (p > 0.05). The genotype frequencies of T/T, T/C, C/C at locus +1312 were 94.6%, 2.7% and 2.7% respectively in the susceptible stock, while 100%, 0% and 0% respectively in the resistant stock. The amino acid change for the mutation at locus +536 A-G was from asparagine to serine, and the predicted homology model of this amino acid variation could affect its function as well as the structural integrity of the domain. In vitro elastase inhibition assay of the protein variants at locus +536 was conducted to explicate the effect of SNP. The increasing concentration of protein (0 mmol/L- 2.93 mmol/L) was incubated with 80 nmol/L elastase where the residual enzyme activity values for rAiSPI (N) with A variant and rAiSPI (S) with G variant were started to reduce from 0.40 to 0.215 and 0.435 to 0.356, respectively. The elastase inhibition ability of rAiSPI (N) variant was significantly higher than that of rAiSPI (S) (p < 0.01). The results suggested that the mutation at locus +536A/A significantly associated with disease resistance of bay scallop would shed light for selective breeding program.

Dietary Exposure to Individual Polybrominated Diphenyl Ether Congeners BDE-47 and BDE-99 Alters Innate Immunity and Disease Susceptibility in Juvenile Chinook Salmon.

Polybrominated diphenyl ethers (PBDEs), used as commercial flame-retardants, are bioaccumulating in threatened Pacific salmon. However, little is known of PBDE effects on critical physiological functions required for optimal health and survival. BDE-47 and BDE-99 are the predominant PBDE congeners found in Chinook salmon collected from the Pacific Northwest. In the present study, both innate immunity (phagocytosis and production of superoxide anion) and pathogen challenge were used to evaluate health and survival in groups of juvenile Chinook salmon exposed orally to either BDE-47 or BDE-99 at environmentally relevant concentrations. Head kidney macrophages from Chinook salmon exposed to BDE-99, but not those exposed to BDE-47, were found to have a reduced ability in vitro to engulf foreign particles. However, both congeners increased the in vitro production of superoxide anion in head kidney macrophages. Salmon exposed to either congener had reduced survival during challenge with the pathogenic marine bacteria Listonella anguillarum. The concentration response curves generated for these end points were nonmonotonic and demonstrated a requirement for using multiple environmentally relevant PBDE concentrations for effect studies. Consequently, predicting risk from toxicity reference values traditionally generated with monotonic concentration responses may underestimate PBDE effect on critical physiological functions required for optimal health and survival in salmon.

The TNFα converting enzyme (TACE) from ayu (Plecoglossus altivelis) exhibits TNFα shedding activity.

TNFα converting enzyme (TACE) is responsible for converting membrane-anchored TNFα to its soluble form in mammalian. However, the function and characteristics of TACE in teleosts is unclear. In this study, we report the cloning of a cDNA sequence of the PaTACE from ayu, Plecoglossus altivelis. PaTACE encodes an 865-aa polypeptide, which is closest to the TACE gene found in pufferfish (Takifugu rubripes). PaTACE mRNA was detected in all the tissues tested, although it was considerably higher in liver, spleen, and brain tissues following infection with Listonella anguillarum. The recombinant region including the PaTACE catalytic domain was used to produce anti-PaTACE IgG. Western blot results revealed two bands for PaTACE from monocytes/macrophages. PNGase F digestion confirmed that the high molecular mass of PaTACE was caused by glycosylation. TACE activity in cell homogenates from ayu monocytes/macrophages increased following L. anguillarum infection. Moreover, PaTACE neutralization led to downregulation of TNFα expression in the supernatant of ayu monocyte/macrophages. Anti-PaTACE IgG also decreased respiratory burst in monocytes/macrophages. In conclusion, we report for the first time the TNFα-converting activity of TACE from a teleost. More investigation is needed to illustrate PaTACE-shedding activity in other immune regulators.

Molecular cloning and expression of a C-type lectin gene from Venerupis philippinarum.

C-type lectins have been demonstrated to play important roles in invertebrate innate immunity by mediating the recognition of pathogens and clearing the micro-invaders. In the present study, a C-type lectin gene (denoted as VpCTL) was identified from Venerupis philippinarum by expressed sequence tag and rapid amplification of cDNA ends approaches. The full-length cDNA of VpCTL consists of 904 nucleotides with an open-reading frame of 456 bp encoding a peptide of 151 amino acids. The deduced amino acid sequence of VpCTL shared high similarity with C-type lectins from other species. The C-type lectin domain and the characteristic EPN and WND motifs were found in VpCTL. The VpCTL mRNA was dominantly expressed in the haemocytes of the V. philippinarum. After Listonella anguillarum challenge, the temporal expression of VpCTL mRNA in haemocytes was increased by 97- and 84-fold at 48 and 96 h, respectively. With high expression level in haemocytes and hepatopancreas, and the up-regulated expression in haemocytes indicted that VpCTL was perhaps involved in the immune responses to L. anguillarum challenge.

Molecular characterization of an IL-1ß gene from ayu, Plecoglossus altivelis.

IL-1ß plays a crucial role as a prototypical proinflammatory cytokine in immune responses and has been shown to affect macrophage functions. However, the effects of putative IL-1ß homologs on fish macrophages are still less known. Here, we cloned the full-length cDNA sequence of IL-1ß (aIL-1ß) gene from ayu, Plecoglossus altivelis. Phylogenetic analysis indicated that aIL-1ß was closest to that of Atlantic salmon (Salmo salar). Real-time quantitative PCR (RT-qPCR) revealed that aIL-1ß transcript was mainly expressed in spleen, head kidney and gill, and dramatically increased in various tissues after Listonella anguillarum infection. Subsequently, aIL-1ß was prokaryotic expressed and purified to prepare anti-aIL-1ß antibody. After L. anguillarum challenge, the aIL-1ß mRNA and protein levels were significantly up-regulated in ayu monocytes/macrophages. Moreover, aIL-1ß neutralization did not change phagocytic capability, but reduced bacterial killing capability in ayu head kidney-derived monocytes/macrophages. Therefore, aIL-1ß may play an important role in immune response of ayu, especially, contributing to bacterial killing of monocytes/macrophages.

Luminal uptake of Vibrio (Listonella) anguillarum by shed enterocytes--a novel early defence strategy in larval fish.

As adhesion and translocation through fish gut enterocytes of the pathogen Vibrio (Listonella) anguillarum are not well investigated, the effective cause of disease and mortality outbreaks in larval sea bass, Dicentrarchus labrax, suffering from vibriosis is unknown. We detected V. anguillarum within the gut of experimentally infected gnotobiotic sea bass larvae using transmission electron microscopy and immunogold labelling. Intact bacteria were observed in close contact with the apical brush border in the gut lumen. Enterocytes contained lysosomes positive for protein A-gold particles suggesting intracellular elimination of bacterial fragments. Shed intestinal cells were regularly visualized in the gut lumen in late stages of exposure. Some of the luminal cells showed invagination and putative engulfment of bacterial structures by pseudopod-like formations. The engulfed structures were positive for protein A-colloidal gold indicating that these structures were V. anguillarum. Immunogold positive thread-like structures secreted by V. anguillarum suggested the presence of outer membrane vesicles (MVs) hypothesizing that MVs are potent transporters of active virulence factors to sea bass gut cells suggestive for a substantial role in biofilm formation and pathogenesis. We put forward the hypothesis that MVs are important in the pathogenesis of V. anguillarum in sea bass larvae.

Association of CfLGBP gene polymorphism with disease susceptibility/resistance of Zhikong scallop (Chlamys farreri) to Listonella anguillarum.

Lipopolysaccharide and ß-1, 3-glucan binding protein (LGBP) is a pattern recognition receptor (PRR) recognizing and binding both LPS and ß-1, 3-glucan, playing important roles in innate immunity. In the present study, the single nucleotide polymorphisms (SNPs) were assessed in LGBP gene from scallop Chlamys farreri (designated CfLGBP), and eight SNPs were found in its potential LPS and glucanase binding motif. The locus +7679 with the transition of G-A, which produced an amino acid substitution at codon 360 from a non polar Glycine to polar Serine, was selected to inspect their association with disease resistance/susceptibility to Listonella anguillarum. Three genotypes G/G, G/A and A/A, were revealed at locus +7679, and their frequencies were 89.7%, 7.7% and 2.6% in the resistant stock, while 63.2%, 34.2% and 2.6% in the susceptible stock, respectively. The frequency of genotypes G/G and G/A were significantly different (P < 0.05) between the two stocks. The pathogen-associated molecular patterns (PAMP) binding activity of two recombinant proteins, rCfLGBP (G) with G variant at locus +7679 and rCfLGBP (S) with A variant at locus +7679, were elucidated by ELISA assay. The binding affinities of both LPS and ß-glucan binding affinity were varied in a dose-dependent manner, where the binding affinity of rCfLGBP (G) was significantly higher than that of rCfLGBP (S) (P < 0.05). The results collectively suggested that the polymorphism of +7679 G/G in CfLGBP possibly enhances the binding activity of LPS and ß-glucan, and was associated to disease resistance of scallop against L. anguillarum, which could be a potential marker applied in future selection of scallop with enhanced resistance to L. anguillarum.

Three ferritin subunits involved in immune defense from bay scallop Argopecten irradians.

Ferritin is a ubiquitous protein that plays an important role in iron storage and iron-withholding strategy of innate immunity. In this study, three genes encoding different ferritin subunits were cloned from bay scallop Argopecten irradians (AiFer1, AiFer2 and AiFer3) by rapid amplification of cDNA ends (RACE) approaches based on the known ESTs. The open reading frames of the three ferritins are of 516 bp, 522 bp and 519 bp, encoding 171,173 and 172 amino acids, respectively. All the AiFers contain a putative Iron Regulatory Element (IRE) in their 5'-untranslated regions. The deduced amino acid sequences of AiFers possess both the ferroxidase center of mammalian H ferritin and the iron nucleation site of mammalian L ferritin. Gene structure study revealed two distinct structured genes encoding a ferritin subunit (AiFer3). Quantitative real-time PCR analysis indicated the significant up-regulation of AiFers in hemocytes after challenged with Listonella anguillarum, though the magnitudes of AiFer1 and AiFer2 were much higher than that of AiFer3. Taken together, these results suggest that AiFers are likely to play roles in both iron storage and innate immune defense against microbial infections.

Phenotypic and genetic diversity of coexisting Listonella anguillarum, Vibrio harveyi and Vibrio chagassi recovered from skin haemorrhages of diseased sand smelt, Atherina boyeri, in the Gulf of Trieste (NE Adriatic Sea).

AIMS: This study identified and characterized coexisting Vibrios associated with haemorrhagic skin lesion bearing sand smelt fishes (Atherina boyeri) in north-eastern Adriatic Sea. METHODS AND RESULTS: Bacteria were isolated from external skin lesions of four samples, and representative morphotypes grown on thiosulfate-citrate-bile salt-sucrose agar were isolated. In total 25 isolates, presumptively assigned to Vibrio genus, were biochemically characterized and were grouped in 10 phenotypic profiles. Phenotypes were heterogeneously distributed among the diseased sand smelt analysed; only one phenotype was recovered from all the samples. Sequencing of 16S rRNA was performed to identify representatives of all phenotypes. Phylogenetic analysis using the neighbour-joining method revealed six isolates clustered within the Vibrio harveyi group, three clustered with known Vibrio chagasii strains and three clustered with Listonella anguillarum. CONCLUSIONS: Vibrios with a broad phenotypic variability were found in the external lesions of diseased A. boyeri. In total three species of Vibrio were identified: V. harveyi showed the wider phenotypical and ribotypical heterogeneity while L. anguillarum shared similar biochemical characteristics with typical strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Previously unreported coexistence of potential pathogenic species colonizing diseased A. boyeri has ecological as well as epidemiological significance.

[Alteration on the expression of ayu coagulation factor X gene upon Listonella anguillarum infection].

Coagulation factor X (FX) plays an important role in the immune response of mammals. In this study, the full length cDNA sequence of the ayu FX gene, 1817 bp in length excluding 3'-polyA tail, was determined for the first time. The sequence contained an open reading frame, which encoded a protein of 453 amino acids with a molecular weight of 5.07×10(4). The predicted protein had motifs typical of animal FX, and its N-terminal 24 residues were the signal peptides. Sequence comparison showed that ayu FX shared 53% amino acid sequence identity with zebrafish FX. In healthy ayu, FX mRNA was expressed mainly in the liver and weakly in the brain and gill. After Listonella anguillarum infection, liver FX transcriptions significantly increased, and peaked at 16 h post infection. The serine protease motif of ayu FX was expressed in Escherichia coli and was subsequently used for antiserum preparation. Western blotting analysis revealed that serum FX significantly increased in bacterially infected ayu fish. In conclusion, the ayu FX gene expression was significant in the progress of bacterial infection, which suggests FX's role in fish immune response.

A novel type III crustin (CrusEs2) identified from Chinese mitten crab Eriocheir sinensis.

Antimicrobial peptides are important effectors in the host innate immune response against microbial invasion. In the present study, the cDNA encoding a crustin (designated CrusEs2) was cloned from Chinese mitten crab Eriocheir sinensis by using EST analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of CrusEs2 was of 1237 bp, containing a 5' untranslated region (UTR) of 12 bp, a 3' UTR of 886 bp with a poly (A) tail, and an open reading frame (ORF) of 339 bp encoding a polypeptide of 112 amino acids with a signal peptide of 19 amino acids. The CrusEs2 contained a typical WAP domain, but lacked the Gly-rich domain of the type II crustin and the Cys-rich region present in both type I and type II crustin, suggesting that CrusEs2 should be classified as a type III crustin. The mRNA transcripts of CrusEs2 could be detected in haemocytes and gill, and its expression level in haemocytes was up-regulated after Listonella anguillarum challenge, while decreased after Micrococcus luteus challenge. The mature peptide coding region of CrusEs2 was cloned into pET-21a+ and expressed in Escherichia coli. The purified recombinant CrusEs2 inhibited the growth of Gram-positive bacteria at MIC of 0.093-0.37 µM. The results indicated that CrusEs2 was involved in immune response of E. sinensis against bacterial challenge.

Identification and characterization of a novel cathelicidin from ayu, Plecoglossus altivelis.

Cathelicidins, one family of antimicrobial peptides, play important roles against infections in animals. In this study, a cDNA sequence coding for cathelicidin was cloned from constructed liver cDNA library of ayu, Plecoglossus altivelis. The deduced ayu cathelicidin (aCATH) has a 20 amino acid residue signal peptide, a conserved cathelin domain of 110 amino acid residues and a mature antimicrobial peptide of 61 amino acid residues. Sequence comparison and phylogenetic tree analysis confirmed aCATH as a distinct member of fish cathelicidins. Real-time quantitative PCR revealed that the aCATH transcripts dramatically increased in various tissues after bacterial infection. Subsequently, aCATH was prokaryotic expressed and purified. Western blot and mass spectrometry revealed that aCATH was cleaved at residue Ile130-Arg131 by human neutrophil elastase to release the mature antimicrobial peptide. The mature peptide of aCATH was chemically synthesized and exhibited potent antimicrobial activity. Thus, aCATH may play an important role in the innate immunity of ayu, and this work enriches our knowledge in fish antimicrobial peptides.

cDNA cloning and mRNA expression of a selenium-dependent glutathione peroxidase from Zhikong scallop Chlamys farreri.

The glutathione peroxidases are essential enzymes of the cellular antioxidant defence system. In the present study, the full-length cDNA sequence encoding an extracellular glutathione peroxidase (designated CfGPx3) was isolated from Zhikong scallop Chlamys farreri. The complete cDNA was of 1194 bp, containing a 5' untranslated region (UTR) of 50 bp, a 3' UTR of 490 bp and an open reading frame (ORF) of 654 bp encoding a polypeptide of 217 amino acids. CfGPx3 possessed all the conserved features critical for the fundamental structure and function of glutathione peroxidase, such as the selenocysteine encoded by stop codon UGA, the GPx signature motif (96LGVPCNQF103) and the active site motif (179WNFEKF184). The high similarity of CfGPx3 with GPx from other organisms indicated that CfGPx3 should be a new member of the glutathione peroxidase family. By fluorescent quantitative real-time PCR, the CfGPx3 mRNA was universally detected in the tissues of haemocytes, gill, gonad, muscle and hepatopancreas with the highest expression in hepatopancreas. After scallops were challenged by Listonella anguillarum, the expression level of CfGPx3 transcript in haemocytes was significantly up-regulated (P<0.05) at 8h post challenge. These results suggested that CfGPx3 was potentially involved in the immune response of scallops and perhaps contributed to the protective effects against oxidative stress.

Disease susceptibility of salmon exposed to polybrominated diphenyl ethers (PBDEs).

The health effects of the flame retardant polybrominated diphenyl ethers (PBDEs) in fish are not well understood. To determine the potential effects of this ubiquitous contaminant class on fish health, juvenile subyearling Chinook salmon (Oncorhynchus tshawytscha) were fed a diet that reflected the PBDE congeners found in the stomach contents of subyearling Chinook salmon collected from the highly urbanized and industrialized lower Willamette River in the Columbia River Basin of North America. The diet, consisting of five PBDE congeners (BDE-47, BDE-99, BDE-100, BDE-153 and BDE-154), was fed to the salmon at 2% of their body weight in food per day for 40 days. Two concentrations of the diet (1x and 10x PBDE) were fed to the salmon. The 1x PBDE diet reflected the concentration of PBDEs (190 ng PBDEs/g food) found in the stomach contents of juvenile subyearling Chinook salmon; the 10x diet was prepared at 10 times that concentration. The fish were then exposed to the marine bacterial pathogen Listonella anguillarum to assess susceptibility to infectious disease. Juvenile Chinook salmon fed the 1x PBDE diet were more susceptible to L. anguillarum than salmon fed the control diet. This suggests that juvenile salmonids in the lower Willamette River exposed to PBDEs may be at greater risk for disease than nonexposed juvenile salmonids. In contrast, salmon that consumed the 10x PBDE diet were not more susceptible to the pathogen than salmon fed the control diet. The mechanisms for the dichotomous results observed in disease susceptibility between salmon fed the 1x and 10x PBDE diets are currently not known but have also been observed in other species exposed to PBDEs with respect to immune function.

An inhibitor kappaB homologue from bay scallop Argopecten irradians.

IkappaB is an important member of NF-kappaB pathway in the innate immune system. In the present study, the full-length cDNA sequence encoding IkappaB protein (designated AiIkappaB) was isolated from bay scallop Argopecten irradians. The complete sequence of AiIkappaB cDNA containing a 5' untranslated region (UTR) of 237 bp, a 3' UTR of 1023 bp with a poly (A) tail, and an open reading frame (ORF) of 1086 bp encoding a polypeptide of 361 amino acids with the predicted molecular weight of 39.9 kDa and theoretical isoelectric point of 4.7. Six ankyrin repeats which were necessary for specific binding to NF-kappaB and two potential phosphorylation sites responsible for IkappaB degradation were identified in the N-terminus of AiIkappaB. No PEST domain but a phosphorylation site motif (S(357)DSD(360)) was present at the C-terminus of AiIkappaB. Predicted three-dimensional structure of AiIkappaB shared high similarity with mammalian IkappaBalpha. Similarity and phylogenetic analysis revealed that AiIkappaB was clustered into IkappaBs from invertebrate. All these typical characteristics indicated that the AiIkappaB should be classified into IkappaB family proteins. Quantitative real-time RT-PCR was employed to assess the mRNA expression of AiIkappaB in various tissues and its temporal expression in haemocytes of scallops challenged with Listonella anguillarum. The mRNA transcript of AiIkappaB could be detected in all the examined tissues with highest expression level in hepatopancreas. Bacteria infection inhibited the transcription level of AiIkappaB. The results suggested the involvement of AiIkappaB in responses against bacterial infection and further highlighted its functional importance in the immune system of A. irradians.

The involvement of suppressors of cytokine signaling 2 (SOCS2) in immune defense responses of Chinese mitten crab Eriocheir sinensis.

The suppressors of cytokine signaling 2 (SOCS2) has been identified as negative feedback inhibitors for various cytokines signaling via the JAK/STAT pathway. In the present studies, the cDNA of Eriocheir sinensis SOCS2 (designated as EsSOCS2) was cloned by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EsSOCS2 was of 2535bp, consisting of an open reading frame (a ORF) of 1071bp encoding a polypeptide of 357 amino acids. The deduced amino acid sequence of EsSOCS2 shared 55-62% similarity with other SOCS2 family members. There were three typical conserved SOCS family domains in EsSOCS2, including an N-terminal ESS formed from a single amphipathic helix, a central SH2 domain with a classic phosphotyrosine (pY) site and a C-terminal SOCS box. The sequence and structural similarity of EsSOCS2 with SOCS2 proteins from other organisms indicated that EsSOCS2 should be a new member of the SOCS2 family. Phylogenetic analysis revealed that EsSOCS2 was clustered with SOCS2 from the other invertebrates, and fell into the group of type II SOCS subfamily as a sister branch to CIS and SOCS2 from vertebrate, suggesting the great divergence of SOCS2 of vertebrate from invertebrate and complex evolution of SOCS2 family members. The mRNA transcript of EsSOCS2 could be detected by semi-quantitative RT-PCR in all examined tissues of healthy crabs, including haemocytes, hepatopancreas, gill, muscle, heart and gonad. The mRNA expression of EsSOCS2 in haemocytes was up-regulated to 3.5-fold at 8h after Listonella anguillarum challenge, 3-fold and 3.5-fold at 4 and 6h, respectively, after Micrococcus luteus challenge. These results collectively suggested that EsSOCS2 could be induced by bacteria challenge, and it was involved in the immune defense responses in E. sinensis.

[Characterization of Listonella anguillarum as the aetiological agent of vibriosis occurred in cultured ayu (Plecoglossus altivelis) in Ninghai country, China].

OBJECTIVE: Ayu (Plecoglossus altivelis) vibriosis threatens ayu aquaculture seriously caused by mass mortality due to severe infections. We characterized the vibriosis pathogen of ayu in Ninghai country. METHODS: A dominant strain was isolated and identified by a series of biochemical and physiological tests. The lethal dose 50% (LD50) was calculated by the modified Karber's method. PCR amplification and sequence analysis were used to further identify the pathogen. RESULTS: LD50 of ayu-H080701 was 1.2 x 10(4) CFU to ayu. PCR amplification showed that the bacterial universal primers for 16S rRNA gene and the specific primers for the metalloprotease (MP) gene of Listonella anguillarum worked. 16S rRNA gene analysis showed that ayu-H080701 shared 99.4%-99.5% nucleotide identical to L. anguillarum isolates, while 94.3% and 91.9% nucleotide identical to L. pelagius and Photobacterium damselae respectively. Metalloprotease analysis showed that ayu- H080701 shared 97.6%-98.8% amino acid sequence identical to L. anguillarum isolates, while lower than 75.6 % to other bacteria. Phylogenetic analysis showed that ayu-H080701 grouped constantly with L. anguillarum isolates. CONCLUSION: The biochemical, physiological tests and sequence analysis all strongly supported the identification of the pathogen causing ayu vibriosis in Ninghai country, China, as an isolate of L. anguillarum.

The genomic structure, alternative splicing and immune response of Chlamys farreri thioester-containing protein.

CfTEP is a member of thioester-containing protein (TEP) family found in Zhikong scallop Chlamys farreri and is involved in innate immunity against invading microbes. In the present study, the genomic DNA of CfTEP was cloned and characterized. The genomic DNA sequence of CfTEP consisted of 40 exons and 39 introns spanning 35kb with all exon-intron junction sequences agreeing with the GT/AG consensus. The genomic organization of CfTEP was similar to human and mouse C3 rather than ciona C3-1 and Drosophila dTEP2. By RT-PCR technique, seven different cDNA variants of CfTEP (designated as CfTEP-A-CfTEP-G) were cloned from scallop gonad. CfTEP-A-CfTEP-F were produced by alternative splicing of six mutually exclusive exons (exons 19-24), respectively, which encoded the highly variable central region. While in CfTEP-G, the deletion of all the six exons introduced a new translation stop site and might trigger nonsense mediated decay (NMD). The mRNA expression and the proportion of the seven CfTEP variant transcripts were examined in the gonad of scallops after bacterial challenge. The fragments containing the highly variable central region of CfTEP were amplified by RT-PCR and a 100 positive clones were sequenced randomly. The expression profiles of the seven CfTEP variants were different and displayed the sex and bacteria dependent manner. In the blank, sea water and Listonella anguillarum challenged subgroups of male scallops, all the transcripts detected were CfTEP-G isoform. In the Micrococcus luteus challenged subgroup, the isoforms expressed and their proportions were CfTEP-F (54%), CfTEP-B (23%), CfTEP-A (10%), CfTEP-C (7%) and CfTEP-E (6%). However, in the gonad of female scallops, only CfTEP-A were found in blank and sea water challenged subgroups. After L. anguillarum or M. luteus challenge, four and five isoforms were detected, respectively, with CfTEP-F isoform being the most one in the both subgroups. These results suggested that the evolution of TEP genes was very complex, and that the diverse CfTEP transcripts generated by alternative splicing played an important role as pattern recognition receptors in the innate immune defense of scallops.

A thioredoxin with antioxidant activity identified from Eriocheir sinensis.

Thioredoxin, with a redox-active disulfide/dithiol in the active site, is the major ubiquitous disulfide reductase responsible for maintaining proteins in their reduced state. In the present study, the cDNA encoding thioredoxin-1 (designated EsTrx1) was cloned from Chinese mitten crab Eriocheir sinensis by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EsTrx1 was of 641 bp, containing a 5' untranslated region (UTR) of 17 bp, a 3' UTR of 306 bp with a poly (A) tail, and an open reading frame (ORF) of 318 bp encoding a polypeptide of 105 amino acids. The high similarity of EsTrx1 with Trx1s from other animals indicated that EsTrx1 should be a new member of the Trx1 sub-family. Quantitative real-time PCR analysis revealed the presence of EsTrx1 transcripts in gill, gonad, hepatopancreas, muscle, heart and haemocytes. The expression of EsTrx1 mRNA in haemocytes was up-regulated after Listonella anguillarum challenge, reached the maximum level at 6h post-stimulation, and then dropped back to the original level gradually. In order to elucidate its biological functions, EsTrx1 was recombined and expressed in E. coli BL21 (DE3). The rEsTrx1 was demonstrated to possess the expected redox activity in enzymatic analysis, and to be more potent than GSH in antioxidant capacity. These results together indicated that EsTrx1 could function as an important antioxidant in a physiological context, and perhaps is involved in the responses to bacterial challenge.