Nile tilapia TRIM39 recruits I3K413 and I3KL45 as adaptors and is involved in the NF-κB pathway.

Tripartite motif (TRIM) proteins play a regulatory function in cancer, cell apoptosis and innate immunity. To understand the role of TRIM39 in Nile tilapia (Oreochromis niloticus), TRIM39 cDNA was isolated. The total length of TRIM39 cDNA was 5025 bp. The deduced OnTRIM39 protein contains 549 amino acids and has conserved domains of the TRIM family, which are the RING, B-box, coiled-coil and PRY-SPRY domains. OnTRIM39 mRNA was widely expressed in various tissues. After challenge with Streptococcus agalactiae and stimulation with polyinosinic polycytidylic acid [poly (I:C)] and lipopolysaccharides (LPS), the amount of OnTRIM39 transcript was changed in various tested tissues. OnTRIM39 overexpression increased NF-κB activity. OnTRIM39 was present in the cytoplasm. Mass spectrometry of proteins pulled down with recombinant OnTRIM39 showed that 250 proteins potentially interact with OnTRIM39. The authors selected I3K4I3 from the 250 candidate proteins to verify its interaction with TRIM39. They also selected I3KL45, a member of the same 14-3-3 protein family, to verify its interaction with TRIM39. The results of pull-down assays showed that OnTRIM39 interacted with both I3K413 and I3KL45. These results contribute to further study of the innate immune mechanism of tilapia.

Isolation, identification, and pathogenic characteristics of Nocardia seriolae in largemouth bass Micropterus salmoides.

The largemouth bass Micropterus salmoides is an important freshwater aquaculture fish in China. Recently, largemouth bass at a fish farm in Guangdong province experienced an outbreak of a serious ulcer disease. As part of the investigations conducted to identify the aetiology and identify potentially effective control measures, we isolated a pathogenic bacterium (NK-1 strain) from the diseased fish. It was identified as Nocardia seriolae through morphological observation, physiological and biochemical analysis, and molecular identification, and its pathogenicity was verified by experimental infection. Pathological changes in the diseased fish included granulomatous lesions in the liver and spleen, destruction of renal tubules, necrosis of intestinal epithelial cells, infiltration of inflammatory cells in the brain, vacuolation of cells, and swelling and cracking of the mitochondria and endoplasmic reticulum. Bacterial detection using qPCR showed that the spleen and intestine were the main organs targeted by N. seriolae. The mortality of largemouth bass experimentally infected with N. seriolae at 21°C was significantly lower than that in fish infected at higher temperatures between 24 and 33°C; there were no significant differences in the levels of mortality at these higher temperatures. The level of mortality of largemouth bass infected with N. seriolae was lowest at a neutral water pH of 7 but increased significantly at higher and lower pH. Of the tested Chinese herbal medicines, Chinese sumac Galla chinensis and Chinese skullcap Scutellaria baicalensis exhibited the best antibacterial effects. This study lays a foundation for the clinical diagnosis and scientific control of ulcer disease in largemouth bass.

TLR1 in Nile tilapia: The conserved receptor cannot interact with MyD88 and TIRAP but can activate NF-κB in vitro.

Toll-like receptors (TLRs) play a critical role in the innate immune response of fish. In this study, we isolated the cDNA sequence of Nile tilapia TLR1 (OnTLR1). The deduced OnTLR1 protein contains a signal peptide, 7 leucine-rich repeats (LRRs), a C-terminal LRR (LRR-CT), a transmembrane region and a highly conserved TIR domain. In healthy Nile tilapia, the OnTLR1 transcript was broadly expressed in all examined tissues, with the highest expression levels in the spleen. After infection with Streptococcus agalactiae, the OnTLR1 transcripts were upregulated in the gill and kidney. After stimulation with polyinosinic-polycytidylic acid (poly(I:C)), the expression levels of OnTLR1 were significantly downregulated in the intestine, whereas OnTLR1 transcripts were significantly upregulated in the kidney. After challenge with lipopolysaccharide (LPS), the expression levels of OnTLR1 were significantly upregulated in the spleen and kidney. The subcellular localization showed that OnTLR1 was expressed in the cytoplasm. TLR1 significantly increased MyD88-dependent NF-κB activity. However, the results of a pull-down assay showed that OnTLR1 did not interact with MyD88 or TIRAP. Binding assays revealed the specificity of OnTLR1 for pathogen-associated molecular patterns (PAMPs) and bacteria that included S. agalactiae, Aeromonas hydrophila and poly(I:C) and LPS. Taken together, these findings suggest that OnTLR1, as a pattern recognition receptor (PRR), might play an important role in the immune response to pathogen invasion.

Structurally diverse genes encode TLR13 in Nile tilapia: The two receptors can recognize Streptococcus 23S RNA and conduct signal transduction through MyD88.

Toll-like receptors (TLRs) play a crucial role in the innate immune system, which is the first line of defence against pathogens and pathogenic products in fish. In the present study, we cloned the full-length cDNA and genome sequences of two TLR13 s (OnTLR13a, OnTLR13b) from Nile tilapia (Oreochromis niloticus). TLR family motifs, i.e., the leucine-rich repeat (LRR) domains and Toll/interleukin (IL)-1 receptor (TIR) domains, were conserved in the putative proteins OnTLR13a and OnTLR13b, with fifteen LRR domains and one TIR domain. Four exons and three introns were identified in the OnTLR13a genome sequence, and three exons and two introns were identified in the OnTLR13b genome sequence. In healthy Nile tilapia tissues, OnTLR13a and OnTLR13b were ubiquitously expressed in all 11 tested tissues/organs. The highest expression levels were observed in the spleen (OnTLR13a) and blood (OnTLR13b), and the lowest expression levels were observed in the liver (OnTLR13a) and stomach (OnTLR13b). The expression level of OnTLR13b at 5.5 days postfertilization (dpf) was significantly higher than that at the other 8 time points (2.5, 3.5, 4.5, 5, 6, 6.5, 7.5 and 8.5 dpf). Upon stimulation with an intraperitoneal injection of 200 µL (107 CFU/mL) Streptococcus agalactiae, the expression levels of OnTLR13a and OnTLR13b were significantly upregulated in the intestine and gill. After cotransfection with MyD88, OnTLR13a significantly increased MyD88-dependent NF-κB activation in 293 T cells. However, OnTLR13b significantly impaired MyD88-dependent NF-κB activation. In addition, TLR13a slightly increased MyD88-dependent AP-1 activation, and TLR13b significantly increased MyD88-dependent AP-1 activation. TLR13a significantly increased MyD88-dependent interferon-ß (IFN-ß) activation, and TLR13b had no effect on MyD88-dependent IFN-ß activation. These findings suggest that although the deduced protein structure of OnTLR13 is evolutionarily conserved between OnTLR13 and other TLR members, its signal transduction function is markedly different. Co-immunoprecipitation (Co-IP) assays showed that both OnTLR13a and OnTLR13b could interact with OnMyD88. RNA pulldown assays showed that TLR13a and TLR13b could combine with the 23S rRNA of S. agalactiae. These results indicate that TLR13a and TLR13b play important roles in the innate immune response against bacterial infection in Nile tilapia.

Identification and characterisation of tumour necrosis factor receptor (TNFR) associated factor 3 from Nile tilapia, Oreochromis niloticus.

In this study, we cloned the complementary (c)DNA sequences of tumour necrosis factor receptor (TNFR)-associated factor 3 (traf3) in Nile tilapia, Oreochromis niloticus. The expression patterns of the traf3 gene were investigated and preliminary functional analyses were performed. In healthy fish, traf3 transcript was broadly expressed in all examined tissues, with the highest expression level in the blood and the lowest in the liver. The traf3 gene reached its highest expression at 8 days post-fertilisation (dpf) during embryonic development. Moreover, we found that expression of traf3 was clearly altered following stimulation with Streptococcus agalactiae in vivo and that traf3 could be induced by lipopolysaccharides (LPS), Poly I: C and S. agalactiae WC1535 in Nile tilapia macrophages. Overexpression in 293T cells showed that Traf3 protein was mainly distributed in the cytoplasm and could significantly increase nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. Taken together, these results implied that traf3 could play important roles in the immune response to pathogen invasion.

Digital gene expression analysis in the liver of ScpB-vaccinated and Streptococcus agalactiae-challenged Nile tilapia.

In recent years, streptococcal diseases have severely threatened the development of tilapia aquaculture, but effective prevention and control methods have not yet been established. To understand the immune responses of vaccinated Nile tilapia (Oreochromis niloticus), digital gene expression (DGE) technology was applied in this study to detect the gene expression profile of the Nile tilapia (O. niloticus) liver in response to ScpB (Streptococcal C5a peptidase from group B Streptococcus, ScpB) vaccination and a Streptococcus agalactiae-challenge. The control and the ScpB-vaccinated Nile tilapia yielded a total of 25,788,734 and 27,088,598 clean reads, respectively. A total of 1234 significant differentially expressed unigenes were detected (P < 0.05), of which 236 were significantly up-regulated, and 269 were significantly down-regulated (P < 0.05, |fold|>2, FDR<0.05). Of the differentially expressed gene, the identified genes which were enriched using databases of GO and KEGG could be categorized into a total of 67 functional groups and were mapped to 153 signaling pathways including 15 immune-related pathways. The differentially expressed genes (TLR1, TLR2, TLR3, TLR5, TLR9, MyD88, C3, IL-1ß, IL-10) were detected in the expression profiles, and this was subsequently verified via quantitative real-time PCR (qPCR). The results of this study can serve as a basis for future research not only on the molecular mechanism of S. agalactiae invasion, but also on the anti-S. agalactiae mechanism in targeted tissues of Nile tilapia.

MHC Class IIB gene polymorphisms associated with resistance/susceptibility to Streptococcus agalactiae in Nile tilapia Oreochromis niloticus.

Genetic variation in the major histocompatibility complex (MHC) Class IIB was tested in Nile tilapia Oreochromis niloticus, and the association between the MHC IIB alleles and disease resistance was also studied. F3 fry offspring (n = 1200) from 12 full-sib families were challenged with Streptococcus agalactiae, which caused significantly different mortalities in different Nile tilapia families (11.00-81.10%). Twenty fry (F1) from each of the 12 families were selected to study the polymorphisms of the MHC Class IIB gene using PCR followed by cloning and sequencing methods. The results showed that the size of the amplified fragment was 770-797 bp. Thirty-seven sequences from 240 individuals revealed 22 different alleles, which belonged to 9 major allele types. Up to 63.58% of nucleotide positions were variable, while the proportion of the amino acid variable positions was up to 68.73%. According to the survival rate of offspring (F3) from 12 full-sib families, we deduced that the alleles Orni-DAB*0107, Orni-DAB*0201 and Orni-DAB*0302 were highly associated with resistance to S. agalactiae, while the allele Orni-DAB*0701 was associated with susceptibility to S. agalactiae. In addition, our previous study found that the allele Orni-DAB*0201 was more frequently distributed in the disease-resistant groups. Therefore, the allele Orni-DAB*0201 could be used as an S. agalactiae resistance-related MHC marker in molecular marker-assisted selective breeding programs for S. agalactiae-resistant Nile tilapia.

MHC class IIA polymorphisms and their association with resistance-susceptibility to Streptococcus agalactiae in Nile tilapia, Oreochromis niloticus.

The association between major histocompatibility complex (MHC) class IIA polymorphisms and the severity of infection by Streptococcus agalactiae was investigated using 40 susceptible and 40 resistant individuals of Nile tilapia Oreochromis niloticus. Twenty-five alleles were identified from 80 individuals, which belong to 22 major allele types. High polymorphism of mhcIIa gene and at least two loci were discovered in O. niloticus. In peptide-binding region (PBR) and non-PBR, the ratio of nonsynonymous substitution (dN) to synonymous substitution (dS) was 1.294 (>1) and 1.240 (>1), suggesting that the loci are evolving under positive balancing selection. Association analysis showed that the allele, orni-daa*0501, was significantly associated with resistance to S. agalactiae, while the alleles, orni-daa*1101, orni-daa*1301, orni-daa*1401 and orni-daa*1201, were associated with susceptibility to S. agalactiae. To confirm these correlations, another independent challenge experiment was performed in the Huizhou population of the O. niloticus. The frequency distribution showed that the orni-daa*1101 allele was significantly more frequent in the Huizhou-Susceptible group (HZ-SG) than in the Huizhou-Resistant group (HZ-RG) (P < 0.05), which was consistent with the first challenge. However, orni-daa*0501 did not present in HZ-SG and HZ-RG and the distribution frequencies of the orni-daa*1201, orni-daa*1301 and orni-daa*1401 alleles were not significantly more frequent in HZ-SG than in HZ-RG. These results indicate that the orni-daa*1101 allele confers susceptibility to S. agalactia infection. These results suggest that the diversity of exon 2 of mcaIIa alleles could be used to explore the association between disease susceptibility or resistance and the multiformity of mcaIIa and to achieve the molecular-assisted selection of O. niloticus with enhanced disease resistance.

Molecular characterization and function analysis of three RIG-I-like receptor signaling pathway genes (MDA5, LGP2 and MAVS) in Oreochromis niloticus.

The recognition of microbial pathogens, which is mediated by pattern recognition receptors (PRRs), is critical to the initiation of innate immune responses. In the present study, we isolated the full-length cDNA and genomic DNA sequences of the MDA5, LGP2 and MAVS genes in Nile tilapia, termed OnMDA5, OnLGP2 and OnMAVS. The OnMDA5 gene encodes 974 amino acids and contains two caspase-associated recruitment domains (CARDs), a DExDc domain (DExD/H box-containing domain), a HELICc (helicase superfamily C-terminal) domain and a C-terminal regulatory domain (RD). The OnLGP2 gene encodes 679 amino acids and contains a DExDc, a HELICc and an RD. The OnMAVS gene encodes 556 amino acids and contains a CARD, a proline-rich domain, a transmembrane helix domain and a putative TRAF2-binding motif (269PVQDT273). Phylogenetic analyses showed that all three genes from Nile tilapia were clustered together with their counterparts from other teleost fishes. Real-time PCR analyses showed that all three genes were constitutively expressed in all examined tissues in Nile tilapia. OnMDA5 presented the highest expression level in the blood and the lowest expression level in the liver, while OnMAVS presented the highest expression level in the kidney. The highest expression level of OnLGP2 was detected in the liver. An examination of the expression patterns of these RIG-I-like receptors (RLRs) during embryonic development showed that the highest expression levels of OnMDA5 occurred at 2 days postfertilization (dpf), and the expression significantly decreased from 3 to 8 dpf. The expression levels of OnLGP2 significantly increased from 4 to 8 dpf. The expression levels of OnMAVS mRNA were stable from 2 to 8 dpf. Upon stimulation by intraperitoneal injection of Streptococcus agalactiae, the expression levels of OnMDA5 were first downregulated and then upregulated in the blood, gill and spleen. In the intestine and kidney, the expression of OnMDA5 was first upregulated, then downregulated, and then upregulated again. The expression of OnLGP2 was upregulated in the kidney and intestine, and the expression of OnMAVS was upregulated in the spleen. Overexpression of OnMAVS increased NF-κB activation in 293 T cells (p < 0.05), and after cotransfection with OnMDA5, the OnMAVS-dependent NF-κB activation was slightly increased (p > 0.05), after cotransfection with OnLGP2, the OnMAVS-dependent NF-κB activation was significantly decreased (p < 0.05). These findings suggest that, although the deduced protein structure of OnMDA5 is evolutionarily conserved with the structures of other RLR members, its signal transduction function is markedly different. The results also suggest that OnLGP2 has a negative regulatory effect on the OnMAVS gene. OnMDA5 and OnMAVS were uniformly distributed throughout the cytoplasm in 293 T cells, whereas OnLGP2 was distributed throughout the cytoplasm and nucleus. These results are helpful for clarifying the innate immune response against bacterial infection in Nile tilapia.

Molecular characterization, expression and functional analysis of NOD1, NOD2 and NLRC3 in Nile tilapia (Oreochromis niloticus).

The nucleotide-binding oligomerization domain proteins NOD1, NOD2 and NLRC3 are cytoplasmic pattern recognition receptors (PRRs) of the Nod-like receptor (NLR) family. In the present study, the Nile tilapia (Oreochromis niloticus) NOD1 (ntNOD1), NOD2 (ntNOD2) and NLRC3 (ntNLRC3) genes were cloned and characterized. The full-length ntNOD1, ntNOD2 and ntNLRC3 genes were 3924, 3886 and 4574 bp, encoding 941, 986 and 1130 amino acids, respectively. The three Nod-like receptors have a NACHT domain and a C-terminal leucine-rich repeat (LRR) domain. In addition, ntNOD1 and ntNOD2 have a N-terminal CARD domain (ntNOD2 has two). Phylogenetic analysis showed that the three NLRs are highly conserved. Tissue expression analysis of the three receptors revealed that the highest mRNA and protein levels of ntNOD1, ntNOD2 and ntNLRC3 were in the spleen. The expression patterns of NLRs during embryonic development showed that the expression levels of ntNOD2 and ntNLRC3 significantly increased from 2 to 8 days post-fertilization (dpf). The expression levels of ntNOD1 significantly increased from 2 to 6 dpf, decreased at 7 dpf and then increased at 8 dpf. Upon stimulation with an intraperitoneal injection of Streptococcus agalactiae, expression levels of the ntNOD1, ntNOD2 and ntNLRC3 mRNA and protein were clearly altered in the blood, spleen, kidney, intestine and gill. Furthermore, after cotransfection with an NF-κB reporter plasmid, NF-κB activation in ntNOD1-overexpressing 293T cells significantly increased compared with that in control cells, before or after i-EDPA-stimulation. By contrast, compared with control, ntNOD2 and ntNLRC3 had no effect on NF-κB activation in 293T cells, when their potential ligands were not stimulated. However, after MDP-stimulation, ntNOD2 and ntNLRC3 overexpression increased NF-κB activation in 293T cells. NOD1 and NLRC3 were uniformly distributed throughout the cytoplasm in 293T cells, whereas NOD2 was distributed throughout the cytoplasm and nucleus. Our results indicate that the three Nod-like receptors are functionally conserved and may play pivotal roles in defense against pathogens such as Streptococcus agalactiae.

[Expression and clinical significance of nerve growth factor in primary liver cancer].

OBJECTIVE: To determine the expression of nerve growth factor (NGF) in hepatic tissues and serum of patients with primary liver cancer (PLC), and to investigate the relationship of serum NGF levels with clinicopathological features of PLC. METHODS: Hepatocellular carcinoma (HCC) samples and patient-matched tumor-adjacent liver samples were collected from 26 PLC patients to assess the mRNA and protein expressions of NGF by reverse transcription-PCR, western blotting (b-actin normalized), and immunohistochemistry. In addition, serum samples were collected from 40 PLC patients, 40 liver cirrhosis patients, 40 chronic hepatitis patients (including hepatitis B or C virus infections), and 30 healthy (normal) controls. The serum levels of NGF were measured by enzyme-linked immunosorbent assay. Intergroup differences were assessed by the t-test, and correlation with sex, age, presence of cirrhosis, tumor size, and TNM classification were assessed by the Kruskal-Wallis H test followed the Mann-Whitney U test. RESULTS: HCC tissues showed higher mRNA and protein expressions of NGF than the corresponding tumor-adjacent non-HCC tissues. Hepatic NGF expression was mainly localized to the tumor cell cytoplasm. Serum NGF expression was significantly higher in PLC patients (33.86+/-16.11 pg/ml) and cirrhosis patients (20.57+/-9.73 pg/ml) than in normal controls (11.13+/-6.12 pg/ml) and chronic hepatitis patients (13.20+/-6.23 pg/ml) (P less than 0.01). Furthermore, when the PLC patients were stratified according to tumor size and TNM stage, the serum NGF level was found to be significantly higher in patients with tumors more than 5 cm (vs. less than 5 cm; U=83.000, P=0.002) or of TNM stage III/IV (vs. stage I/II; U=103.500, P=0.009). CONCLUSION: Elevated expression of NGF in liver cancer tissues and serum of PLC patients is related with tumor size and TNM staging. These findings suggest that NGF may play a role in HCC tumorigenesis and/or that serum NGF may represent a prognostic marker of PLC.

Major histocompatibility complex class IIA and IIB genes of Nile tilapia Oreochromis niloticus: genomic structure, molecular polymorphism and expression patterns.

Major histocompatibility complex (MHC) is a large genomic region characterized by extremely high polymorphism, and it plays an important role in the immune response of vertebrates. In the present study, we isolated MHC class II genes from Nile tilapia in order to investigate the immune mechanism in tilapia and develop better strategies for disease prevention. Moreover, we cloned the full-length cDNA sequences of MHC IIA and IIB from Nile tilapia by the RACE approach. In addition, the genomic structure, molecular polymorphism and expression patterns of MHC II genes in Nile tilapia were also examined. Compared with that of other teleosts, Nile tilapia MHC class IIA contained four exons and three introns. The deduced amino acid sequence of the MHC IIA molecule shared 25.4-64.5% similarity with those of other teleosts and mammals. Six exons and five introns were identified from Nile tilapia MHC IIB, and the deduced amino acid sequence shared 26.9-74.7% similarity with those of other teleosts and mammals. All the characteristic features of MHC class II chain structure could be identified in the deduced sequences of MHC IIA and IIB molecules, including the leader peptide, α1/ß1 and α2/ß2 domains, connecting peptide and transmembrane and cytoplasmic regions, as well as conserved cysteines and N-glycosylation site. A total of 12 MHC IIA alleles were identified from six individuals. Four alleles originating from a single individual suggested that at least four MHC IIA loci existed. Moreover, 10 MHC IIB alleles were identified, among which four were detected in a single individual, suggesting that at least four MHC IIB loci existed. The expression of MHC IIA and IIB at the mRNA level in 10 types of normal tissues was determined using quantitative real-time PCR analysis. The highest expression level was detected in stomach and gill, whereas the lowest expression was detected in muscle and brain. Furthermore, MHC IIA and IIB were probably two candidate immune molecules involved in the resistance against streptococcosis, because their expression was significantly up-regulated in gill, kidney, intestine and spleen after the intraperitoneal injection of Streptococcus agalactiae.