Abstract:Mammalian cylindromatosis (CYLD) has been identified as a tumor suppressor and participates in innate immune signaling transduction in various cell types through negative regulation of NF-κB activation by deubiquitinating TRAF6 and NEMO. To investigate the role of CYLD in the immune response of large yellow croaker (Larimichthys crocea), the cDNA sequence of CYLD was cloned and identified, named LcCYLD. Gene expression profile was detected by real-time fluorescence quantitative PCR. Then the recombinant plasmid pTurboGFP-CYLD was constructed for subcellular localization and transfected into HEK293T cells. For further understanding of the function of CYLD in innate immune, the recombinant overexpression vector pcDNA3.1-CYLD was constructed and transfected into cells, and the activation capability of NF-κB, proinflammatory factors TNF-α and IL-1β were detected. Sequence analysis showed that the ORF of LcCYLD contained 2 754 bp, encoding 917 amino acids. The putative LcCYLD protein contained three conservative N-terminal CAP_GLY domain, a phosphorylation region, and a typical C-terminal UCH domain. Multiple alignments showed that CYLD was highly conserved among the analyzed species. Phylogenetic analysis showed that LcCYLD was clustered with bony fish and closely related to striped bass (Morone saxatilis). Gene expression analysis indicated that LcCYLD expressed in most examined tissues with the most predominant expression in the brain, followed by blood and intestine. However, the expression levels in other tissues are very weak. LPS and poly I:C stimulation significantly induced the transcriptional expression of LcCYLD. Subcellular localization showed that LcCYLD expressed both in cytoplasm and nuclease. Overexpression of LcCYLD could significantly inhibit the immuno-activation of NF-κB and proinflammation of cytokines TNF-α and IL-1β after LPS and poly I:C challenge. These findings suggested that LcCYLD could negatively regulate NF-κB activation. The present study might be helpful for better understanding the function of LcCYLD in innate immune signaling transduction of L. crocea.

Abstract:Serine protease (SP) is a kind of important and widely distributed proteolytic enzyme, which participates in a series of important physiological processes, including digestion, blood coagulation, embryonic development and immune response. But there are only a few preliminary reports about SP in crustaceans. Shrimp farming is constantly threatened by diseases. Further research on shrimp immune defense mechanism will provide clues for finding new ideas of disease resistance. In this study, the full-length cDNA sequence of a novel serine protease homologs (SPH) gene (Lv-SPH, GenBank accession number: KR020739) was cloned from hemocytes of Litopenaeus vannamei according to our previous transcriptome results. The sequence characteristics were analyzed by bioinformatics method. Tissue distribution and expression profile in response to virus infection were discussed by real-time fluorescence quantitative PCR. Bioinformatics analysis showed that the total length of the gene was 1 249 bp, the open reading frame (ORF ) region was 1 005 bp encoding 334 aa with 156 bp 5′-UTR and 88 bp 3′-UTR. The deduced amino acid sequence contained a signal peptide sequence with 16 amino acids at the amino end. SMART analysis showed that Lv-SPH contained a clip domain and a highly conserved SP domain (Tryp-SPc), which had two of the three active catalytic sites (His and Asp), and the third one is Gly. Lv-SPH was expressed in different tissues of L. vannamei. The main expression was in hemocyte, followed by hepatopancrease, heart and intestine. The lowest expression level was in muscle. Real time fluorescence quantitative PCR analysis showed that white spot syndrome virus (WSSV) could induce the up-regulated expression of Lv-SPH significantly. The relative expression of Lv-SPH increased significantly at 24 h and reached the highest level at 48 h, which was about three times that of the control group. Lv-SPH has typical characteristics of SPH family members and has tissue expression specificity. WSSV can significantly induce the up-regulated expression of Lv-SPH gene, indicating that it may be involved in the immune response process of L. vannamei induced by WSSV. The results provide a reference for further exploring shrimp innate immune regulation mechanism, and have potential application value in shrimp healthy culture and disease control.

Abstract:Shrimp iridescent virus disease is an acute and infectious disease that has widely broken out in crustaceans in China in recent years. Its pathogen is Decapoda iridescent virus 1 (DIV1) that belongs to a new genus Decapodiridovirus of family Iridoriridae. The DIV1 has a high transmission risk, wide host range, and extensive mortality, leading to serious economic losses in cultured shrimp in China. Some research has been reported on the etiology, pathology, epidemiology, detection and diagnosis of shrimp iridescent disease, but little is known about the molecular mechanism of viral infection and the host responses to DIV1. Here, we provided an overview of the latest research progresses on the discovery, classification status, morphological characteristics. and chemical composition and physicochemical properties of DIV1, and introduced the genomic information, pathogenicity, host ranges, route of transmission, detection and diagnostic technology, as well as the shrimp's responses. The disease prevention and control strategies and future studies on Decapoda iridescent virus 1 were also suggested. This information will promote understanding of the pathogenic mechanism of DIV1, which will benefit virological research and the effective control of shrimp iridescent virus disease.

Abstract:To investigate the cause of an outbreak of unknown disease occurred in cultured largemouth bass (Micropterus salmoides) in a reservoir in Fujian Province, with a cumulative mortality of over 30% in March 2021, the dominant bacterial colonies were isolated from the liver, spleen and kidney tissues of affected fish, the pathogenic bacteria was identified by artificial infection experiments, and its species was identified by physicochemical characteristics, 16S rDNA sequences, and mass spectrometry analyses. Further, virulence gene detection, drug sensitivity research and histopathological observation were carried out. A predominant bacteria strain Yersinia ruckeri was isolated from the spleen of the inffected M. salmoides. A set of virulence genes including YrP1⁺ - YhlA^{+ -} YhlB⁺ - invF^- - traC^- - traL^- - traN^- were detected by PCR from in the isolated strain, with a LD₅₀ of 3.8×10⁵ CFU per fish by intraperitoneal injection. The antibiotic sensitivity test showed that the pathogen was sensitive to enrofloxacin, doxycycline hydrochloride, flumequine, neomycin sulfate and florfenicol. Histopathological observation found that Y. ruckeri infection caused significant damages to liver, spleen, kidney and other organs of M. salmoides. The main pathologic lesions showed obvious degeneration, necrosis and infiltration of the inflammation cells. It could be included that the current research is the first report of cultured M. salmoides infected by Y. ruckeri, which will be helpful for the early diagnosis and better prevention of the disease in cultured M. psalmodies.

Abstract:In order to explore the role of rhamnose-binding lectin (RBL) in the non-specific cell defense of bony fishes against pathogen infection. The head kidney monocytes/macrophages from Nile tilapia (Oreochromis niloticus) were isolated to conduct a bacterial stimulation experiment in vitro. The expression of Onrbl-1 was significantly up-regulated following challenges with two important pathogens, Streptococcus agalactiae and Aeromonas hydrophila. Meanwhile, OnRBL-1 recombinant protein could participate in the regulation of the expression of pathogen-induced cell inflammatory factors by quantitative real-time PCR (qRT-PCR), including significantly inhibiting the expression of il-6, il-8 and tnf-α, and promoting the expression of il-10 and tgf-β. In addition, the results showed that OnRBL-1 could promote the phagocytosis, enhance the level of respiratory burst and the release of reactive oxygen species in monocytes/macrophages through flow cytometry analysis. Taken together, the results indicated that OnRBL-1 plays an important regulatory role in the non-specific cellular defense of monocytes/macrophages from O. niloticus. This study provides a reference for exploring the function of RBL-1 in host defense against pathogen infection, which helps to explain the action mechanism of bony fish RBL in the defense against pathogen infection and improve the basic theoretical system of its participation in immune response, with great significance in science.

Abstract:To explore the pathogenic mechanism of nocardiosis in Channa argus, we studied the pathogenicity and drug sensitivity of the pathogen and the immune resistance of C. argus by isolation and identification of the pathogen, histopathology and gene expression analysis. The results showed that the diseased C. argus was mainly infected with a pathogen strain SDAT 0011. The strain SDAT 0011 was identified as Nocardia seriolae using the morphologic structure and staining observation, PCR amplification of species-specific primers, sequence analysis of 16S rRNA gene, and physiological and biochemical tests. The challenge experiments showed that the isolated strains were pathogenic on C. argus and the mortality rate of the 1 × 10⁵ − 1 × 10⁸ CFU/mL injection groups was 100%. In addition, the diseased C. argus exhibited numerous marked white nodules (round/ovoid), ranging from 0.1 to 0.2 cm in diameter on internal organs, especially the spleen, kidney, and liver. Histopathological observation and analysis showed that the structure of chronic granuloma was visible, with many lymphocytes, damaged or dead cells in the center. The antibiotic susceptibility assays of the strain SDAT 0011 showed that the strain was sensitive to rifampin, but resistant to penicillin, cefradine, and ampicillin, etc. Furthermore, the expression levels of toll-like receptors 2 gene (TLR2), toll-like receptors 13 gene (TLR13), and C-C chemokine receptor type 9 gene (CCR9) after infection were analyzed by real-time fluorescence quantitative PCR (qRT-PCR). In our study, following N. seriolae challenge, TLR2 and TLR13 were significantly up-regulated, while CCR9 was significantly down-regulated at all examined time points in the spleen and head kidney. These results implied that some genes of the toll-like receptor signaling pathway and the chemokine signaling pathway have been activated in the early stages of infection to resist Nocardia infection. The results of this study will lay a foundation for the treatment of N, seriolae and studying it pathogenesis..

Abstract:To accurately identify the pathogen of Siniperca chuatsi, the experiments were based on morphological comparisons, combined with parasitic characterization and molecular phylogenetic methods to carry out the study and observe the ultrastructure. The results showed: fusiform or dumb-bell white sporangia, about 1.9-6.8 mm in length, were observed in the fins, skin, oral cavity, gills, the outer and the inner layer of the operculum of the diseased fishes; endospores of (9.8±1.8) (7.3-13.8) μm in diameter; the ring-like cytoplasm and circular refractive bodies of about (6.5±1.2) (3.9-8.3) μm in diameter. It was identified as Dermocystidium. The measured values of this species were different from those of other Dermocystidium and were in general agreement with those of the undetermined species (HB) parasitizing the gills of Siniperca chuatsi; it was parasitized by similar sites as Dermocystidium from Guangdong and Henan provinces (GD and HN) and Dermocystidium sp. (HB). Molecular sequence alignment and phylogenetic results showed that this species shared over 99% similarity with Dermocystidium sp. (HB and HN), Dermocystidium anguillae , D. fennicum, and D. salmonis in SSU rDNA sequences. Phylogenetic analysis indicated that D. anguillae sampled from Guangdong (GD) and Henan province (HN) branched closely with the present species, then D. salmonis, D. anguillae, and D. fennicum. We concluded that Dermocystidium sp. infecting Siniperca sp. are conspecific and we revised the records of the D. anguillae (GD and HN) isolates of Siniperca chuatsi; we named Dermocystidium in this study together with the isolates (HB, GD and HN) as Dermocystidium sinipercae sp. n.. The intraspecific morphological variation of Dermocystidium sinipercae sp. n. may be related to the sampling time and developmental environment; spore proliferation and differentiation processes were recorded and described in ultrastructural observations. This study confirms that Dermocystidium sinipercae sp. n. has been distributed in many parts of China, emphasizing the urgent need to implement quarantine measures for the origin of Siniperca chuatsi.

Abstract:Vitamin C-based carbon dots are a new class of nanomaterials with antibacterial properties, but their antibacterial activities against aquatic pathogenic bacteria have not been verified. For this reason, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of V_C-CDs against 12 aquatic pathogenic bacteria such as Aeromonas hydrophila, Vibrio parahaemolyticus, V. splendens and Nocardia sp. were determined using 3-fold serial dilutions. The bacteriostatic kinetic curves of the 12 aquatic pathogens were determined. In addition, based on the potential biocompatibility of nano materials, we used MTT method to detect the cytotoxicity of V_C-CDs on CIK cells. Danio rerio embryos were used as test objects to determine the embryotoxicity after exposure to V_C-CDs. The results showed that V_C-CDs had significant inhibitory effect on 12 aquatic pathogenic bacteria, and the minimum inhibitory concentrations ranged from 20.6 to 61.7 μg/mL, basically reaching the same antibacterial effect as the antibiotic florfenicol. The survival rate of zebrafish embryos and CIK cells was almost 100% in the range of MIC. This study shows that V_C-CDs have good bactericidal and antibacterial effects on the main pathogenic bacteria in aquaculture, and have good biocompatibility, which signifies the potential as a promising alternative to antibiotics in the prevention and control of bacterial diseases in aquaculture.

Abstract:Dendritic cells are the outposts of the immune system, which link innate immunity with adaptive immunity, and thus are considered as the most powerful antigen-presenting cells in vivo. To further investigate the functions of CD8α and CD207 in grass carp (Ctenopharyngodon idella) dendritic cells (DCs), the recombinant eukaryotic expression plasmids of CD8α and CD207 genes were constructed and successfully expressed in DCs of C. idella. The open reading frames (ORFs) of CD8α and CD207 were obtained from DCs of C. idella by RT-PCR and inserted into recombinant eukaryotic expression plasmid pcDNA3.1, respectively, to construct recombinant eukaryotic expression plasmids pcDNA3.1-CD8α and pcDNA3.1-CD207; after the sequencing was confirmed to be correct, the recombinant eukaryotic expression plasmids were transfected into DCs of C. idella using liposome method, and the expressions of CD8α and CD207 were verified by cell immunofluorescence technology and Western blot. The results showed that in the Western blot assay, the CD8α and CD207 proteins were expressed normally and significantly overexpressed after transfection of the recombinant eukaryotic expression plasmid, and the size was consistent with that of the predicted result. Cellular immunofluorescence showed that the CD8α and CD207 proteins were mainly expressed on the cell membrane of DCs. Hence, DCs of C. idella exhibit immunophenotypes and functions that are conserved in their mammalian counterparts. The recombinant eukaryotic expression plasmids of pcDNA3.1-CD8α and pcDNA3.1-CD207 were successfully constructed, which laid a foundation for further research on the action mechanism of CD8α and CD207 genes in DCs. Carrying out studies on the immunophenotype of DCs will play a positive role in analyzing the DCs-mediated immune regulation mechanism of C. idella. In the cultivation process, C. idella are easily infected by pathogens and induce inflammatory reactions. The development of DCs-based immunotherapy can provide new ideas for the prevention and control of C. idella related diseases, which has a wide application prospect in reducing diseases and enhancing the immune efficacy of vaccines for teleost fish.

Abstract:To investigate whether probiotics can regulate nonspecific immune enzyme activities, blood biochemical indexes and expression of related genes of Cynoglossus semilaevis under ammonia nitrogen stress, Bacillus subtilis Y2 as a probiotic was fed to C. semilaevis, and then ammonia nitrogen stress was applied to C. semilaevis. During the stress, Y2 was continuously fed and the related indexes were monitored. The results of macroscopical growth indexes showed that the body weight and length of the C. semilaevis in the ammonia nitrogen stress groups were lower than those of the non-ammonia nitrogen stress groups, and the figures for the Y2 groups were higher than those of the blank groups both with and without ammonia nitrogen stress. The results of immune enzyme assays showed that the activities of catalase (CAT), acid phosphatase (ACP), peroxidase (POD) and alkaline phosphatase (AKP) of C. semilaevis were increased after ammonia nitrogen stress; the activities of ACP, CAT and POD in the two groups fed with Y2 strain with and without ammonia nitrogen stress were higher than those in the control groups. The results of blood biochemical indexes showed that the contents of albumin (ALB) and globulin (GLB) in serum of C. semilaevis in the non-ammonia nitrogen stress Y2 group were slightly higher than those in the control group, the albumin/globulin ratio (A/G) was higher, and the contents of triglyceride (TG) and cholesterol (CHO) had little difference; under ammonia nitrogen stress, the contents of ALB and CHO in the serum of C. semilaevis in the control group was decreased, the content of GLB was slightly increased, the total protein (TP) and total fat in serum were decreased, and the A/G was decreased. However, Y2 could increase the content of ALB, TG and CHO, under the condition of ammonia nitrogen stress resulting in the increase of the A/G. At the same time, the content of malondialdehyde (MDA), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum was significantly increased under ammonia nitrogen stress. Y2 could reduce the content of MDA, AST and ALT both with and without ammonia nitrogen stress. The results of the related gene expression showed that the expression levels of heat shock protein gene (HSP70) and hemoglobin α 1 gene (HB-α1) in intestine, liver, muscle and gill of C. semilaevis were increased under ammonia nitrogen stress, and the expression of HSP70 in Y2 group was higher than that in control group, with the increase most obvious in liver tissue. On the contrary, Hb-α1expression in Y2 group decreased, especially in liver and muscle tissue. In addition, the expression of growth factor gene (IGF) in intestine, liver, muscle and gill of C. semilaevis was decreased under ammonia nitrogen stress, but the expression of IGF in both Y2 groups was higher than that in control groups both with and without ammonia nitrogen stress. In conclusion, Y2 can alleviate the effects of ammonia nitrogen stress on the immune ability, blood biochemical indexs, oxygen transport, stress ability and growth of C. semilaevis, reducing the negative effects of ammonia stress on C. semilaevis. It has a good prospect for application in the aquaculture industry.

Abstract:Toll-like receptor gene family is a class of conserved pattern recognition receptors, which plays an essential role in innate immunity providing efficient defense against invading microbial pathogens. Epinephelus akaara is one of the most important commercial marine fishes, and the main aquaculture industry in China is distributed along the coast of Fujian. The species is also considered a good model for studying immunity. Although TLRs have been extensively characterized in both invertebrates and vertebrates, a comprehensive analysis of TLRs in E. akaara is lacking. This research aims to study the systematic evolution, chromosome distribution, and expression regulation patterns of the Toll-like receptor genes in different tissues of E. akaara, based on the published genome and transcriptomic data of E. akaara, this study analyzed the phylogenesis, chromosome distribution, and expression regulation patterns of Toll-like receptor gene family in E. akaara tissues using bioinformatics methods including BLAST, phylogeny and synteny. The results showed that a total of 17 TLR genes were identified in E. akaara, which were divided into 5 subfamilies (TLR1, TLR3, TLR5, TLR7 and TLR11) and distributed on 11 of the 24 chromosomes. The 17 TLRs showed different expression patterns in different tissues. EaTLR1-2, EaTLR2-2, and EaTLR13-2 were mainly highly expressed in the spleen. EaTLR5M was highly expressed in the kidney, spleen, gills, and heart, but lowly expressed in other tissues. EaTLR5S was highly expressed in the kidney, liver, and spleen, but with low expression in other tissues. EaTLR18-1 and EaTLR18-2 were mainly expressed in the heart. EaTLR8 was highly expressed in all tissues except muscle and liver. EaTLR1-1, EaTLR2-1a, EaTLR2-1b, EaTLR3, EaTLR7, EaTLR9, EaTLR13-1, EaTLR21, and EaTLR22 were mainly highly expressed in the brain, spleen, kidney, and gill. In addition, EaTLR18-1 and EaTLR18-2 were both located on chromosome 9 with highly similar expression patterns. EaTLR2-1a and EaTLR2-1b were located on chromosome 20 and their expression patterns were also highly similar. In addition, EaTLR5M and EaTLR5S, which had high LRR domain similarity, were located on chromosome 14, and had high similarity in expression levels in different tissues. On the contrary, EaTLR7 and EaTLR8, which were located on chromosome 18, and EaTLR2-2 and EaTLR3, which were located on chromosome 4, had different expression patterns. These suggested that copies of TLR genes in the same chromosome may have similar expression patterns or functions. This work provided reference data for studying the evolution of the Toll-like receptor system in fish, and laid a foundation for further research on the function of Toll-like receptor genes in E. akaara.

Abstract:Vibrio alginolyticus is one of the main pathogens that causes mass mortality in aquatic animals and infects humans. To reduce the application of antibiotics, alternative therapies have been proposed. One of the promising possibilities is to control diseases through the use of lytic bacteriophages. As few current bacteriophages against V. alginolyticus have been reported, the main objective of this study is to develop effective bacteriophages for controlling pathogenic bacteria. This study describes the isolation and characterization of lytic bacteriophages against a V. alginolyticus strain (VAHN1). The bacteriophages were isolated from Hainan shrimp pond and Fujian marine products by double-layer agar culture method, and V. alginolyticus VAHN1 was used as the host strain for bacteriophage isolation. The phages were classified and identified by transmission electron microscope (TEM), restriction endonuclease and phylogenetic tree analysis.The physiological and biochemical characteristics of the bacteriophages were determined, including optimal multiplicity of infection (MOI), lysis spectrum detection, pH stability, thermal stability, resistance to UV light, and sensitivity to chloroform and ether. In addition, the effect of bacteriostasis were also measured. The results showed that 2 lytic bacteriophages against V. alginolyticus were isolated, named as VAP9 and VAP21. The plaques of VAP9 and VAP21 were neat and transparent, with a diameter of 1.5-2 mm. The nucleotides of the phage VAP9 and VAP21 were all dsDNA, and all their heads were shown as icosahedral shape with about 55 nm and 65 nm diameter respectively under TEM. The tail of the phage VAP9 was approximately 65-70 nm in length and 15 nm in width, while the tail of the phage VAP21 was about 75-80 nm in length and 18 nm in width. The 2 bacteriophages are grouped under the Myoviridae family. The phage VAP9 and VAP21 had good tolerance to different physical and chemical environment. The survival rate of the 2 bacteriophages was greater than 43% at 60 °C for 2 h. The optimal pH of VAP9 was 6-8 and VAP21 was 7-11. The 2 bacteriophages tolerated peracetic acid in universal bactericidal concentration. The phages VAP9 and VAP21 were insensitive to chloroform and ether, with a certain resistance to UV light. The optimal MOIs of both VAP9 and VAP21 were 0.001. The cocktail of VAP9 and VAP21 was able to infect 95.2% (157 strains among 165 strains) of the V. alginolyticus strains and 50% (3 strains among 6 strains) of the V. parahaemolyticus strains used in this study. And they could not infect other species of tested bacteria except V. alginolyticus and V. parahaemolyticus. The phages VAP9 and VAP21 could effectively inhibit the growth of V. alginolyticus VAHN1 and have the same inhibitory trend on VAHN1, but the VAP21 strain had a stronger bacteriostatic effect than that of the VAP9 strain. Moreover, the inhibitory effect involved the cocktail of 2 bacteriophages on V. alginolyticus were better than that of a single phage. Alignment with the sequences of the conserved protein amino acid sequences of the phage VAP9 and VAP21 on National Center for Biotechnology Information (NCBI), it was showed that the 2 bacteriophages had low homology with other bacteriophages. Furthermore, phylogenetic and genome analysis revealed that the phages VAP9 and VAP21 have low homology with other bacteriophages and may be 2 novel Myoviridae bacteriophages that infect bacteria related Vibrio spp. This study did not only enrich the species resources of bacteriophages against V. alginolyticus, but it also laid a theoretical foundation for the development and application of Vibrio bacteriophages as microecological antimicrobial agents.

Abstract:Cryptocaryon irritans is a parasitic ciliate causing ‘white spot disease’ on marine teleosts that often results in huge financial losses to mariculture. So far, there is no safe and effective method to control the parasite infection. Calmodulin (CaM), as a sensor for Ca²⁺ signaling, regulates cell movement, cell division and invasion of some protozoan parasites to their hosts. To understand the role of calmodulin in the growth and development of C. irritans, the calmodulin gene (cicam) was cloned from the C. irritans trophozoite cDNA library, and the open reading frame (ORF) was synthesized after codon optimization. The recombinant plasmid pGEX-4T-1/cicam was constructed, then transformed into Escherichia coli and induced its recombinant expression with ZYM-5052 self-inducing medium. The recombinant protein rCiCaM was purified by glutathione agarose gel and thrombin, and obtain polyclonal antibodies by immunizing with the mouse BALB/c strain. The expression of cicam and its encoded protein CiCaM in each stage of the worm was examined by reverse transcription PCR and immunoblotting assay, respectively. The localization of CiCaM in the larvae was examined by indirect fluorescent antibody assay. The activity of recombinant protein rCiCaM binding recombinant actin-depolymerizing factor was initially investigated by blot overlay assay. The results showed that the open reading frame (ORF) of CiCaM was 450 bp, which encoded a polypeptide of 16.9 ku, consisting of 149 amino acids; the molecular masses of GST-rCiCaM and rCiCaM were 43 ku and 16.9 ku respectively, which corresponded with the predicted ones; the CiCaM gene expressed in all developmental stages of C. irritans, and the molecular mass of the native CiCaM corresponded to the predicted value; the CiCaM distributed all over the cytosol of C. irritans theronts, especially abundant around the four macro nuclei and the peripheral area of cytostome; rCiCaM could interact with rCiADF2 in a Ca2+-dependent way. This study enriched the knowledge of molecular biology of the pathogen C. irritans, which would provide a reference for the prevention and control of cryptocaryonosis.

Abstract:Complement and coagulation cascade are important components of innate immunity, in which complement protein 5a (complement component 5a, C5a), complement protein 5a receptor (C5a receptor, C5aR) and coagulation factor II (blood coagulation factor II, FII) play a key role in dealing with virus infection. In order to study their protein expression and interaction in Ctenopharyngodon idella infected with grass carp reovirus (GCRV), the prokaryotic expression system of C5a and FII protein was constructed, the C5aR-KLH conjugate against C5aR protein was constructed, and the protein was purified. Japanese big-eared rabbits were immunized to prepare polyclonal antibodies against the three kinds of proteins. The expression and interaction of the three proteins were detected by western blot, Co-immunoprecipitation (Co-IP) and pulldown test. Western blot results showed that C5a and C5aR proteins were expressed in liver, spleen, kidney, head kidney, intestine, gill and muscle of C. idella. FII protein was expressed in liver, spleen and intestine, but not in kidney, head kidney, gill and muscle. Expression of C5a, C5aR and FII proteins in liver tissues of C. idella infected with GCRV showed an upward trend with the progression of the disease. Co-IP results showed that C5a, C5aR and FII proteins interacted with each other after GCRV treatment. Pull down results showed that 28 candidate proteins such as C3 and RIG-I were identified by C5a pull down, and 24 candidate proteins such as transaldolase and macroglobulin were identified by C5aR pull down. This study provides a basis for further exploring the interaction and regulation of C5a, C5aR and FII, and for further exploring the mechanism of the three associated proteins in the complement and coagulation cascade system responding to GCRV infection.

Abstract:To elucidate the tissue tropism and spreading routes of Haliotid herpesvirus 1 (HaHV-1) in susceptible Haliotis diversicolor aquatilis after infection, an in situ loop-mediated isothermal amplification (LAMP) method for HaHV-1 was developed in this study based on LAMP and in situ hybridization techniques. The developed in situ LAMP method was then applied on pedal nerve branches, mantle, hepatopancreas and gill of specimens collected across the experimental infection process. For the establishment of the HaHV-1 in situ LAMP detection method, we firstly optimized LAMP primers for specific detection of HaHV-1, improving the specificity and stability of the LAMP reaction on slides. We then optimized the time for color development (about 60 min) on pedal nerve branches, which was identified as the most suitable target for pathological investigation and in situ LAMP detection. Finally, the HaHV-1 in situ LAMP method was established after color development with immunoenzymatic labeling technique. The results showed that the incorporation of the designed loop primers yielded stabile results during LAMP amplification. The developed in situ LAMP detection method was applied on major tissue collected at 24、36、48、60 and 72 h across the experimental infection process. The in situ LAMP signals were firstly detected at 36 h on pedal nerve branches, and at 48 h on mantles after infection. The viral signals detected at mantle were confined to the peripheral nerves scattered throughout the mantle. Finally, in situ LAMP signals were identified occasionally in connective tissue of hepatopancreas of samples collected at the end of the experimental infection. The viral infection signals were always accompanied by tissue lesions and infiltrated haemocytes infected with HaHV-1. The in situ LAMP method developed in this study achieved the goal of rapid detection and definite diagnosis of HaHV-1, and is helpful for investigating the tissue tropism and pathological characteristics in different susceptible species. The detection method constitutes a potentially valuable tool for the diagnosis of HaHV-1 infection, and studies associated with tissue tropism, invasion pathways and pathogenic mechanism of HaHV-1.

Abstract:To analyze the effect of the Cyprinus carpio Slc15a2 gene on the immune response mechanism during Aeromonas hydrophila infestation, this experiment prepared rat-derived Slc15a2-1 and Slc15a2-2 polyclonal antibodies, and changes in protein expression of Slc15a2-1 and Slc15a2-2 in C. carpio sera following A. hydrophila infection was detected by enzyme linked immunosorbent assay (ELISA). The results showed that ① compared with the blank control the maximum protein expression levels of the two copies after infection were increased by 4.81 and 2.48 times, respectively. The protein expression of Slc15a2-2 was consistently higher from 0 h to 48 h compared to that of Slc15a2-1. ② Overall, there was a similar expression trend between the two copies of Slc15a2, with both increasing, then decreasing, then increasing and then decreasing again. However, Slc15a2-1 reached its first expression peak at 3 h, while Slc15a2-2 reached its first expression peak at 6 h. ③ Slc15a2-1 and Slc15a2-2 protein expression showed complementary expression trends during the four periods from 6 h to 24 h. That is, the expression of one copy decreased significantly and the expression of the other copy increased significantly during the same period, but the overall expression level remained high. The results indicate that the two polyclonal antibodies prepared in this experiment have high affinity and specificity, and the overall similar expression trend between the two copies after infection with A. hydrophila suggests that they may have similar gene functions. Slc15a2-2 had significantly higher expression between 0 h and 48 h than Slc15a2-1, possibly as the major gene in the copy. The two copies showed complementary expression from 6 h to 24 h, and the co-expression was maintained at a high level, enabling an effective response in the face of pathogen attack. This study may provide some basis for insight into the regulatory immune response mechanism of C. carpio Slc15a2 gene.