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.

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.

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.

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.