Development of real-time recombinase polymerase amplification (RPA) and RPA combined with lateral flow dipstick (LFD) assays for the rapid and sensitive detection of cyprinid herpesvirus 3.

In this issue, we established rapid, cost-effective, and simple detection methods including recombines polymerase amplification with lateral flow dipstick (RPA-LFD) and real-time RPA for cyprinid herpesvirus 3(CyHV-3), and evaluated their sensitivity, specificity, and applicability, the real-time RPA method could achieve sensitive diagnosis of CyHV-3 within 1.3 copies per reaction, respectively. The real-time RPA method is 10-fold more sensitive than RPA-LFD method. The exact number of CyHV-3 can be calculated in each sample by real-time RPA. The sera from koi also can be tested in these methods. In addition, no cross-reaction was observed with other related pathogens, including carp oedema virus (CEV), spring viraemia of carp virus (SVCV), cyprinid herpesvirus 1(CyHV-1), cyprinid herpesvirus 2(CyHV-2), type I grass carp reovirus (GCRV-I), type II GCRV (GCRV-II), type III GCRV (GCRV-III), and Aeromonas hydrophila.

Transcriptome analysis of Tetrahymena thermophila response to exposure with dihydroartemisinin.

Dihydroartemisinin (DHA) is a derivative of artemisinin and is toxic to parasites. We used the Tetrahymena thermophila (T. thermophila) as a model to explore DHA toxicity. Results showed that low concentration of DHA (20 µmol/L) promoted cell proliferation, whereas high concentrations of DHA (40-1280 µmol/L) inhibited that. Appearance of nucleus was pycnosis by laser scanning confocal microscope. DHA significantly elevated activities of SOD and GSH-Px (P < 0.01) and MDA was markedly increased at high level but decreased at low level (P < 0.01). Further results of transcriptome in T. thermophila treated with different concentration DHA group (0, 20, 160 µmol/L) showed that differentially expressed genes (DEGs) were involved in oxidation-reduction and metabolism of exogenous substances indicated oxidative stress stimulation. Kyoto Encyclopedia of Genes and Genomes showed that DEGs were involved in the cytochrome P450-mediated metabolism of exogenous substances, glutathione metabolism and ABC transport. Remarkably, DNA replication was significantly enriched in low concentration DHA, energy metabolism related pathways and necrotic process were considerably enriched in high concentration DHA. The results of RT-qPCR of 13 DEGs were the same as that of transcriptome, in which the expression of GST and GPx family genes were significantly altered after exposed to high-DHA group. DHA induced oxidative stress damage through disturbing with energy. However, detoxification pathways in T. thermophila to resist oxidative damage and cell alleviated low concentration DHA stress by regulating antioxidant enzyme. This study provides good practice on pharmacological mechanism of artemisinin-based drugs in antiparasitic.

Oral delivery of Bacillus subtilis spores expressing Clonorchis sinensis paramyosin protects grass carp from cercaria infection.

Clonorchis sinensis (C. sinensis), an important fishborne zoonotic parasite threatening public health, is of major socioeconomic importance in epidemic areas. Effective strategies are still urgently expected to prevent against C. sinensis infection. In the present study, paramyosin of C. sinensis (CsPmy) was stably and abundantly expressed on the surface of Bacillus subtilis spores. The recombinant spores (B.s-CotC-CsPmy) were incorporated in the basal pellets diet in three different dosages (1 × 105, 1 × 108, 1 × 1011 CFU/g pellets) and orally administrated to grass carp (Ctenopharyngodon idella). The immune responses and intestinal microbiota in the treated grass carp were investigated. Results showed that specific anti-CsPmy IgM levels in sera, skin mucus, bile, and intestinal mucus, as well as mRNA levels of IgM and IgZ in the spleen and head kidney, were significantly increased in B.s-CotC-CsPmy-1011 group. Besides, transcripts levels of IL-8 and TNF-αin the spleen and head kidney were also significantly elevated than the control groups. Moreover, mRNA levels of tight junction proteins in the intestines of B.s-CotC-CsPmy-1011 group increased. Potential pathogenetic bacteria with lower abundance and higher abundances of candidate probiotics and bacteria associated with digestion in 1 × 1011 CFU/g B.s-CotC-CsPmy spores administrated fishes could be detected compared with control group. The amount of metacercaria in per gram fish flesh was statistically decreased in 1 × 1011 CFU/g B.s-CotC-CsPmy spores orally immunized group. Our work demonstrated that B. subtilis spores presenting CsPmy on the surface could be a promising effective, safe, and needle-free candidate vaccine against C. sinensis infection for grass carp.

Berberine Influences Blood Glucose via Modulating the Gut Microbiome in Grass Carp.

Berberine (BBR), an isoquinoline alkaloid, is a major pharmacological component of the Chinese herb Coptis chinensis, which has been listed in the Chinese Fisheries Pharmacopeia as a common drug for the control of bacterial fish diseases. However, BBR is poorly absorbed into the systemic circulation but is significantly accumulated in the intestine. It is difficult to explain the mechanism of clinical effects of BBR based on systemic genes and pathways; it has been proved that the function of BBR in mammals is associated with the host metabolic phenotypes mediated by the structural modulation of gut microbiota. The mechanism of pharmacological effects of BBR in fish remains unclear. Here, we fed grass carp (Ctenopharyngodon idellus) a diet supplemented with BBR at a dose of 30 mg/Kg body weight daily and compared them with grass carp fed a regular fish feed diet. Biochemical analysis revealed that fish fed BBR had significantly reduced serum glucose, total cholesterol (TC), and triglyceride (TG) levels, and increased TC (p < 0.05) and TG (p < 0.01) levels in the liver. Deep amplicon sequencing of the V4 region of 16S rRNA genes of the gut microbiota revealed: (i) the composition of gut microbiota after BBR feeding was more diverse than that in the control group; (ii)before fish were fed BBR, the enriched operational taxonomic units (OTUs) mainly belonged to Firmicutes while most enriched OTUs came from Proteobacteria, Planctomycetes, Bacteroidetes, and Firmicutes during BBR feeding and after BBR feeding stopped; (iii) the ratio of Firmicutes to Bacteroidetes was significantly decreased in fish fed BBR. Spearman's rank correlation showed that 32 berberine-OTUs were significantly negative correlated with glucose (p < 0.05). It indicates that BBR may affect the levels of serum glucose by the structural modulation of gut microbiota. Our results provide insight into the effect of BBR on fish metabolism and gut microbiomes, which would be beneficial for the fish welfare.

Oral delivery of Bacillus subtilis spores expressing cysteine protease of Clonorchis sinensis to grass carp (Ctenopharyngodon idellus): Induces immune responses and has no damage on liver and intestine function.

Clonorchis sinensis (C. sinensis) is a fish-borne trematode. Human can be infected by ingestion of C. sinensis metacercariae parasitized in grass carp (Ctenopharyngodon idella). For induction of effective oral immune responses, spores of Bacillus subtilis (B. subtilis) WB600 were utilized as vehicle to delivery CsCP (cysteine protease of C. sinensis) cooperated with CotC (B.s-CotC-CP), one of coat proteins, to the gastrointestinal tract. After routine culture of 8-12 h in LB medium, B. subtilis containing CotC-CsCP was transferred into the sporulation culture medium. SDS-PAGE, western blotting and the growth curve indicated that the best sporulation time of recombinant WB600 was 24-30 h at 37 °C with continuous shaking (250 rpm). Grass carp were fed with three levels of B.s-CotC-CP (1 × 106, 1 × 107, and 1 × 108 CFU g-1) incorporated in the basal pellets diet. The commercial pellets or supplemented with spores just expressing CotC (1 × 107 CFU g-1) were served as control diet. Our results showed that grass carp orally immunized with the feed-based B.s-CotC-CP developed a strong specific immune response with significantly (P < 0.05) higher levels of IgM in samples of serum, bile, mucus of surface and intestinal compared to the control groups. Abundant colonization spores expressing CsCP were found in hindgut that is conducive to absorption and presentation of antigen. Moreover, B. subtilis spores appeared to show no sign of toxicity or damage in grass carp. Our cercariae challenge experiments suggested that oral administration of spores expressing CsCP could develop an effective protection against C. sinensis in fish body. Therefore, this study demonstrated that the feed-based recombinant spores could trigger high levels of mucosal and humoral immunity, and would be a promising candidate vaccine against C. sinensis metacercariae formation in freshwater fish.

TAK1-binding proteins (TAB1 and TAB2) in grass carp (Ctenopharyngodon idella): identification, characterization, and expression analysis after infection with Ichthyophthirius multifiliis.

Transforming growth factor-ß activated kinase-1 (TAK1) is a key regulatory molecule in toll-like receptor (TLR), interleukin-1 (IL-1), and tumor necrosis factor (TNF) signaling pathways. The activation of TAK1 is specifically regulated by two TAK1-binding proteins, TAB1 and TAB2. However, the roles of TAB1 and TAB2 in fish have not been reported to date. In the present study, TAB1 (CiTAB1) and TAB2 (CiTAB2) in grass carp (Ctenopharyngodon idella) were identified and characterized, and their expression profiles were analyzed after fish were infected with the pathogenic ciliate Ichthyophthirius multifiliis. The full-length CiTAB1 cDNA is 1949 bp long with an open reading frame (ORF) of 1497 bp that encodes a putative protein of 498 amino acids containing a typical PP2Cc domain. The full-length CiTAB2 cDNA is 2967 bp long and contains an ORF of 2178 bp encoding a putative protein of 725 amino acids. Protein structure analysis revealed that CiTAB2 consists of three main structural domains: an N-terminal CUE domain, a coiled-coil domain, and a C-terminal ZnF domain. Multiple sequence alignment showed that CiTAB1 and CiTAB2 share high sequence identity with other known TAB1 and TAB2 proteins, and several conserved phosphorylation sites and an O-GlcNAc site were deduced in CiTAB1. Phylogenetic tree analysis demonstrated that CiTAB1 and CiTAB2 have the closest evolutionary relationship with TAB1 and TAB2 of Danio rerio, respectively. CiTAB1 and CiTAB2 were both widely expressed in all examined tissues with the highest levels in the heart and liver, respectively. After infection with I. multifiliis, the expressions of CiTAB1 and CiTAB2 were both significantly up-regulated in all tested tissues at most time points, which indicates that these proteins may be involved in the host immune response against I. multifiliis infection.

Expression profiles of toll-like receptors in channel catfish (Ictalurus punctatus) after infection with Ichthyophthirius multifiliis.

Toll-like receptors (TLRs) play a crucial role in the innate immune system, but to date the roles of fish TLRs in response to parasitic infection are still poorly understood. In the present study, we used channel catfish (Ictalurus punctatus) and the ciliate parasite Ichthyophthirius multifiliis as a model to investigate whether and which fish TLRs play important roles in the immune response against parasitic pathogens by detecting the expression profiles of a complete set of TLRs in catfish at different time points after infection with I. multifiliis. The expression profiles of TLR1 and TLR2 were similar, and both were significantly up-regulated in the skin and head kidney at most time points after infection. Furthermore, the expression of TLR2 was also up-regulated in the gill and spleen. TLR9 was induced in the skin and gill, whereas TLR21 was induced in the head kidney and spleen after infection. For TLR19, significant up-regulation was observed in the skin and gill, but significant down-regulation was detected in the head kidney and spleen. In contrast to TLR19, TLR25 was significantly up-regulated in the head kidney and spleen at some time points. No significant changes were observed for the rest of the TLRs at most time points. The results indicated that some TLRs may play essential roles in catfish defense against I. multifiliis infection.

Grass carp (Ctenopharyngodon idella) TRAF6 and TAK1: molecular cloning and expression analysis after Ichthyophthirius multifiliis infection.

Ichthyophthirius multifiliis, a pathogenic ciliate parasite, infects almost all freshwater fish species and causes significant economic losses. Tumor necrosis factor receptor-associated factor 6 (TRAF6) and transforming growth factor-ß-activated kinase 1 (TAK1) are two important signaling molecules involved in toll-like receptor (TLR) signal transduction. To date, the roles of TRAF6 and TAK1 in host defense against fish parasites are still poorly understood. In the present study, TRAF6 (CiTRAF6) and TAK1 (CiTAK1) were identified from grass carp (Ctenopharyngodon idella). The full-length cDNA sequence of CiTRAF6 (2250 bp) includes an open reading frame (ORF) of 1629 bp, which shows a high similarity to that of Cyprinus carpio TRAF6 and encodes a putative protein of 542 amino acids containing one RING domain, two zinc fingers, one coiled-coil region, and one MATH domain. The full-length CiTAK1 cDNA sequence is 2768 bp and includes an ORF of 1626 bp that encodes a putative protein of 541 amino acids containing a conserved serine/threonine protein kinase catalytic domain and a coiled-coil region. Phylogenetic analysis showed that CiTRAF6 and CiTAK1 were clustered with TRAF6 and TAK1 of other teleosts, respectively. CiTRAF6 and CiTAK1 were both constitutively expressed in all examined tissues but with varied expression levels. The highest expressions of CiTRAF6 and CiTAK1 were in the head kidney and spleen, respectively. The expression profiles of CiTRAF6 and CiTAK1 were detected in grass carp after I. multifiliis infection. Expressions of both genes were significantly up-regulated in the skin, gill, head kidney, and spleen at most time points after infection, indicating that CiTRAF6 and CiTAK1 may play essential roles in grass carp defense against I. multifiliis.

Genotype and host range analysis of infectious spleen and kidney necrosis virus (ISKNV).

Infectious spleen and kidney necrosis virus (ISKNV) is the causative agent of a disease causing high mortality in mandarin fish, Siniperca chuatsi. In this study, complete major capsid protein (MCP) genes of nine ISKNV isolates were sequenced and compared with other known megalocytiviruses to evaluate genetic variation and host range of the viruses. Comparison of nucleotide sequences of MCP gene revealed 92.6-100% identity among nine ISKNV isolates. A phylogenetic tree revealed that 33 megalocytiviruses were divided into three genotypes, and there was a strong host species signal in three genotypes: for genotype I, the host was mainly marine fish; for genotype II, the host was freshwater fish; and for genotype III, the host was mainly flatfish. Nine ISKNV isolates belonged to genotype I or genotype II, suggesting mandarin fish may be a mixing vessel host for megalocytivirus.

[Inverse PCR amplification of the complete major capsid protein gene of lymphocystis disease virus isolated from Rachycentron canadum and the phylogenetic analysis of the virus].

The major capsid protein of lymphocystis disease virus isolated from Rachycentron canadum (LCDV-rc) was amplified and analysed. The 457bp DNA core fragment was amplified with the degenerate primers designed according to the conserved sequences of MCP gene of iridoviruses, then the flaking sequences adjacent to the core region were amplified by inverse PCR, and the complete sequence was obtained by combining all of them. The open reading frame of the gene is 1380bp in length, encoding a putative protein of 459 aa with molecular weight 51.12 kD and pI 6.87. Constructing the phylogenetic tree for comparing the MCP amino acid of iridoviruses, the results indicated that LCDV-rc is most homologous to the other Lymphocystis viruses and all of them constitute a branch. Accordingly LCDV-rc is identified as Lymphocystivirus.

[Relationships among planktons DNA sequence diversity, water quality and fish diseases in Siniperca chuatsi ponds].

By using random amplified polymorphic DNA (RAPD) technique, this paper studied the alpha-diversity of plankton communities and its relationships with water quality and fish diseases in 7 Siniperca chuatsi ponds, as well as the effects of stocking density and a new culture model on the diversity and water quality. The results showed that there was a significant negative correlation between the DNA sequence diversity of plankton communities and water quality index, and high stocking density decreased the DNA sequence diversity and increased the water quality index. The new culture model with short culture period, low stocking density and high feeding stuff input had a greater damage on the water environment. Hierarchical cluster analysis indicated that there existed similarities in the DNA sequences of plankton communities and the physicochemical properties of water bodies in the ponds with fish diseases, which provided a possibility to predict the diseases occurrence in Siniperca chuatsi ponds.