Guo-Xing Nie

Toll-like receptor recognition of bacteria in fish: ligand specificity and signal pathways.

Pattern recognition receptors (PRRs) recognize the conserved molecular structure of pathogens and trigger the signaling pathways that activate immune cells in response to pathogen infection. Toll-like receptors (TLRs) are the first and best characterized innate immune receptors. To date, at least 20 TLR types (TLR1, 2, 3, 4, 5M, 5S, 7, 8, 9, 13, 14, 18, 19, 20, 21, 22, 23, 24, 25, and 26) have been found in more than a dozen of fish species. However, of the TLRs identified in fish, direct evidence of ligand specificity has only been shown for TLR2, TLR3, TLR5M, TLR5S, TLR9, TLR21, and TLR22. Some studies have suggested that TLR2, TLR5M, TLR5S, TLR9, and TLR21 could specifically recognize PAMPs from bacteria. In addition, other TLRs including TLR1, TLR4, TLR14, TLR18, and TLR25 may also be sensors of bacteria. TLR signaling pathways in fish exhibit some particular features different from that in mammals. In this review, the ligand specificity and signal pathways of TLRs that recognize bacteria in fish are summarized. References for further studies on the specificity for recognizing bacteria using TLRs and the following reactions triggered are discussed. In-depth studies should be continuously performed to identify the ligand specificity of all TLRs in fish, particularly non-mammalian TLRs, and their signaling pathways. The discovery of TLRs and their functions will contribute to the understanding of disease resistance mechanisms in fish and provide new insights for drug intervention to manipulate immune responses.

The transcriptomic response to copper exposure by the gill tissue of Japanese scallops (Mizuhopecten yessoensis) using deep-sequencing technology

The bivalve Mizuhopecten yessoensis has been greatly impacted by marine pollutants in northern China. To elucidate the toxicological mechanism of copper exposure on the immune system, we investigated differentially expressed genes (DEGs) and transcript abundance in M.xa0yessoensis gill tissue using the deep-sequencing platform Illumina HiSeq™ 2000. In total, 1312 and 2237 genes were identified as significantly up- or down-regulated, respectively. In addition, significant enrichment analysis identified 9 GO terms and 38 pathways involved in the response to copper exposure. The analysis of immune-related transcripts revealed a complex repertoire of innate recognition receptors, including toll-like receptors, NOD-like receptors and RIG-like receptors. Downstream pathway effectors, such as apoptotic, lysosomal and C-type lectin transcripts, were also analyzed. These results will provide a resource for subsequent gene expression studies regarding heavy metal exposure and the identification of copper-sensitive biomarkers to monitor the aquaculture of M.xa0yessoensis.

The transcriptomic response to copper exposure in the digestive gland of Japanese scallops (Mizuhopecten yessoensis)

The present study was conducted to elucidate the effects of copper exposure on the immune system and lipid metabolism of the Japanese scallop, Mizuhopecten yessoensis. Transcriptional levels of differentially expressed genes (DEGs)in M. yessoensis digestive gland tissue were analyzed using the deep-sequencing platform Illumina HiSeq™ 2000. In total, 841 and 877 genes were identified as significantly up- or down-regulated, respectively. In addition, significant enrichment analysis identified 3 gene ontology terms and 15 pathways involved in the response to copper exposure. Analysis of transcripts related to the immune response revealed a complex pattern of innate recognition receptors, including toll-like receptors, NOD-like receptors and downstream pathway effectors, including those involved in apoptosis. Furthermore, genomic analysis revealed that genes involved in extracellular matrix (ECM)-receptor interactions were enriched in Cu-exposed scallop glands. These results will provide a resource for subsequent gene expression studies regarding heavy metal exposure and the identification of copper-sensitive biomarkers for the aquaculture of M. yessoensis.

Expression patterns of Toll-like receptors in natural triploid Carassius auratus after infection with Aeromonas hydrophila.

Toll-like receptors (TLRs) play the important roles in the innate immune system. In the present study, we have cloned fragments of 13 members of TLR family in natural triploid Qihe crucian carp (C. auratus) and investigated the expression profiles of these TLRs in spleen and head kidney at different time points after A. hydrophila infection. The results showed that the expressions of certain TLRs including TLR4, TLR5, and TLR22 were significantly up-regulated after infection, and the expression levels of TLR5 and TLR22 were the highest in spleen with 52.56-fold and 28.14-fold increase, respectively, whereas the other TLRs were down-regulated or no significant changes were observed compared with the control at most time points. These findings suggested that three TLRs (TLR4, TLR5, and TLR22) may play important roles in the immune responses of C. auratus to A. hydrophila infection. Additionally, the expression of most TLRs was significantly up-regulated at 6h post infection, which implied that the immune response of C. auratus reached the highest level at this time point. This work will facilitate our comprehensive understanding for the functions of TLRs in the process of bacterial infection in C. auratus and provide new insights for developing preventive and therapeutic measures against A. hydrophila infection.

Immune effects of the vaccine of live attenuated Aeromonas hydrophila screened by rifampicin on common carp (Cyprinus carpio L)

Aeromonas hydrophila, as a strong Gram-negative bacterium, can infect a wide range of freshwater fish, including common carp Cyprinus carpio, and cause the huge economic loss. To create the effective vaccine is the best way to control the outbreak of the disease caused by A. hydrophila. In this study, a live attenuated A. hydrophila strain, XX1LA, was screened from the pathogenic A. hydrophila strain XX1 cultured on medium containing the antibiotic rifampicin, which was used as a live attenuated vaccine candidate. The immune protection of XX1LA against the pathogen A. hydrophila in common carp was evaluated by the relative percent survival (RPS), the specific IgM antibody titers, serum lysozyme activity and the expression profiles of multiple immune-related genes at the different time points following immunization. The results showed that the variable up-regulations of the immune-related genes, such as the pro-inflammatory cytokine IL-1β, the chemokine IL-10 and IgM, were observed in spleen and liver of common carp injected in the vaccines with the formalin-killed A. hydrophila (FKA) and the live attenuated XX1LA. Specific antibody to A. hydrophila was found to gradually increase during 28 days post-vaccination (dpv), and the RPS (83.7%) in fish vaccinated with XX1LA, was significant higher than that (37.2%) in fish vaccinated with FKA (P<0.05) on Day 28 after challenged by pathogen. It was demonstrated that the remarkable immune protection presented in the group vaccinated with XX1LA. During the late stage of 4-week immunization phase, compared with FKA and the control, specific IgM antibody titers significantly increased (P<0.05) in the XX1LA group. The activity of the lysozyme in serum indicated no significant change among three groups. In summary, the live attenuated bacterial vaccine XX1LA, screened in this study, indicates the better protect effect on common carp against A. hydrophila, which can be applied in aquaculture of common carp to prevent from the disease outbreak in the future.

Identification and expression of the laboratory of genetics and physiology 2 gene in common carp Cyprinus carpio

In this study, a laboratory of genetics and physiology 2 gene (lgp2) from common carp Cyprinus carpio was isolated and characterized. The full-length complementary (c)DNA of lgp2 was 3061u2009bp and encoded a polypeptide of 680 amino acids, with an estimated molecular mass of 77 341·2u2009Da and a predicted isoelectric point of 6·53. The predicted protein included four main overlapping structural domains: a conserved restriction domain of bacterial type III restriction enzyme, a DEAD-DEAH box helicase domain, a helicase super family C-terminal domain and a regulatory domain. Real-time quantitative polymerase chain reaction (PCR) showed widespread expression of lgp2, mitochondrial antiviral signalling protein (mavs) and interferon transcription factor 3 (irf3) in tissues of nine organs. lgp2, mavs and irf3 expression levels were significantly induced in all examined organs by infection with koi herpesvirus (KHV). lgp2, mavs and irf3 messenger (m)RNA levels were significantly up-regulated in vivo after KHV infection, and lgp2 transcripts were also significantly enhanced in vitro after stimulation with synthetic, double-stranded RNA polyinosinic polycytidylic [poly(I:C)]. These findings suggest that lgp2 is an inducible protein involved in the innate immune defence against KHV in C. carpio. These results provide the basis for further research into the role and mechanisms of lgp2 in fishes.

Rhodococcus agglutinans sp. nov., an actinobacterium isolated from a soil sample

AbstractnA Gram-positive, aerobic, non-motile and non-spore forming strain, designated CFH S0262T, was isolated from a soil sample collected from Catba island in Halong Bay, Vietnam. A polyphasic approach was used to study the taxonomic position of this new isolate. Phylogenetic analyses based on 16S rRNA gene sequence showed that strain CFH S0262T belongs to the genus Rhodococcus and clustered with Rhodococcus soli DSD51WT, Rhodococcus hoagii NBRC 103062T, Rhodococcus defluvii CallT and Rhodococcus kunmingensis YIM 45607T (98.7, 98.5, 97.9 and 97.6xa0% similarities, respectively). Strain CFH S0262T could grow in the presence of NaCl (0–4xa0%, optimum 0–3xa0%), at pH 6.0–8.0 (optimum, pH 7.0) and at 10–40xa0°C (optimum, 28xa0°C). The predominant menaquinones of strain CFH S0262T were identified as MK-8 (H2) and MK-8 (H4). The major fatty acids (≥10xa0%) were found to be C16:0 and C18:1ω9c. The polar lipids detected were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside, a glycolipid and two unidentified phospholipids. The DNA G+C content was determined to be 71.4xa0mol%. Based on a comparative analysis of phenotypic and genotypic characteristics, in combination with low values of DNA–DNA hybridization between strain CFH S0262T and its closest neighbours, it is proposed that strain CFH S0262T represents a novel species of the genus Rhodococcus, for which the name Rhodococcus agglutinans sp. nov., is proposed, with the type strain CFH S0262T (=CCTCC AB2014297T=KCTC 39118T).

First report of Rahnella aquatilis infection in crucian carp Carassius auratus in China

Rahnella aquatilis infection is rare in aquaculture. Here, a Gram-negative rod-shaped bacterium was isolated from diseased crucian carp Carassius auratus in Xuzhou City, Jiangsu Province, eastern China. The isolate was tentatively named strain KCL-5, and subsequently identified as R. aquatilis by biochemical properties and molecular techniques. The results showed that the isolate KCL-5 was most closely related to the type strain ATCC33071 (= DSM4594) of R. aquatilis, which shared 99.67, 96.26 and 99.58% nucleotide sequence identities for 16S rDNA, gyrB and toxin yhaV genes, respectively. Experimental challenges were conducted which demonstrated pathogenicity of the isolate in crucian carp. Antimicrobial susceptibility testing showed that the isolated strain was susceptible to piperacillin, gentamicin, kanamycin, nalidixic acid, norfloxacin, ofloxacin, azithromycin and erythromycin. To our knowledge, this is the first report on R. aquatilis infection in crucian carp, and the first evidence of pathogenicity in fish.

Nocardia tengchongensis sp. nov., isolated from a soil sample

The taxonomic status was determined of two actinomycetes, designated CFH S0057T and CFH S0065, that were isolated from soil samples collected from an extinct volcano in Tengchong county, Yunnan province, south-west China. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains CFH S0057T and CFH S0065 belong to the genus Nocardia and formed a single clade within this genus. The two isolates were able to grow at 4–45xa0°C, pH 5.0–7.0 and with a NaCl tolerance up to 5.0% (w/v). The whole-cell hydrolysates were rich in meso-diaminopimelic acid, galactose, arabinose and fructose. Mycolic acids were present. Strains CFH S0057T and CFH S0065 exhibited a menaquinone system with MK-8 (H4, ω cyclo), and the major polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and one unidentified phospholipid. Major fatty acids were C16:0, Summed features 3, C18:1ω9c and C18:0 10-methyl (TBSA). The genomic DNA Gxa0+xa0C contents of strains CFH S0057T and CFH S0065 were 65.7 and 66.1xa0mol%, respectively. The combined genotypic, phenotypic and chemotaxonomic results indicated the isolates are considered to represent a novel species of the genus Nocardia, for which the name Nocardia tengchongensis sp. nov. is proposed. The type strain is CFH S0057T (=xa0KCTC 29485Txa0=xa0JCM 30083T).

Nocardia heshunensis sp. nov., an actinomycete isolated from soil

A novel Gram-stain-positive, aerobic, non-motile and acid-fast actinomycete strain, designated CFH S0067T, was isolated from a soil sample collected from Heshun old town in Tengchong, Yunnan province, in south-west PR China. The taxonomic position of strain CFH S0067T was studied in detail using a polyphasic approach. Phylogenetic analysis, based on 16S rRNA gene sequences, revealed that strain CFH S0067T belongs to the genus Nocardia and is closely related to Nocardia concava JCM 12351T (99.3u200a% similarity), forming a separated branch with this type strain. However, the strain shared 96.0u200a% gyrB gene sequence similarity with N. concava JCM 12351T. Furthermore, DNA-DNA hybridization showed 56.5±0.6u200a%u2009DNA relatedness between the novel strain and N. concava JCM 12351T. The whole-cell hydrolysates contained meso-diaminopimelic acid (type IV) and arabinose, galactose, fructose and mannose. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and one unidentified lipid. Strain CFH S0067T contained MK-8 (H4ω-cycl) as the predominant menaquinone. C16u200a:u200a0, summed feature 3 (C16u200a:u200a1ω7c and/or C16u200a:u200a1ω6c), C18u200a:u200a1ω9c and C18u200a:u200a0 10-methyl (TBSA) were the major cellular fatty acids. Mycolic acids were also detected. The G+C content of the genomic DNA was determined to be 66.9u2009mol%. A combination of the low DNA-DNA hybridization values and phenotypic properties demonstrated that strain CFH S0067Tis clearly distinguishable from its most closely related strain, N. concava JCM 12351T. On the basis of this polyphasic study, it is concluded that strain CFH S0067T should be considered to represent a novel species of the genus Nocardia, for which the name Nocardia heshunensis sp. nov. is proposed. The type strain is CFH S0067T (=DSM 46764T=JCM 30085T).