Abstract:To explore the response mechanism of digestion and immune system to “reverse back syndrome” (RBS) of Babylonia areolata, both healthy(control group, CG) and disease samples (early stage of RBS [ERBS], middle stage of RBS [MRBS], and late stage of RBS [ERBS]) were collected, the Vibrio content, the activity of SOD, POD, CAT, ACP, AKP, LZM, pepsin, lipase and amylase in these samples were detected by TCBS plate count methods and enzyme test kit methods. The results showed that the content of Vibrio increased significantly with the development of the disease, and it reached the maximum in LRBS of Babylonia areolata. In MRBS, the activity of POD, AKP and LZM increased, and significantly higher than CG and ERBS; compared with ERBS, the activity of CAT, SOD, ACP, pepsin and lipase decreased; but compared with CG, the CAT and ACP activity increased significantly, and the pepsin and lipase activity decreased significantly, while there was no significant difference in SOD and amylase activity. In LRBS, the activities of digestive enzymes and immune-related enzymes were lower than those in MRBS, and compared with CG, the POD and ACP activity significantly increased, the pepsin, lipase and amylase activity significantly decreased, while there was no significant difference in the CAT, SOD, AKP and LZM activity. In conclusion, the immune system and digestive system are involved in the immune response during the outbreak of Vibrio in the RBS, and the CAT, POD and ACP activity can be used as the detection and evaluation indicators.

Abstract:In August 2018, an infectious disease with high mortality and hemorrhagic symptom occurred in Acipenser baerii in Pengzhou and Qionglai Counties, Sichuan Province, China. Bacterial examination, PCR detection of acipenserid herpesvirus 2 (AciHV-2) and pathological examination of liver and kidney of A. baerii were performed to elucidate the aetiology. Two strains of gram-positives chain-forming cocci were isolated from the diseased A.baerii, and the 16S rRNA gene sequencing analysis by BLAST in GenBank indicated that the two isolates (MN416231 and MN416230) showed a high level of similarity to Streptococcus iniae (more than 99%). A phylogenetic tree based on the 16S rRNA sequences indicated that the two isolates and other S. iniae strains were grouped into a same branch. In addition, specific PCR detection for lctO gene of S. iniae showed that the two isolates were positive. Based on the 16S rRNA gene sequencing analysis and specific PCR detection, two isolates were identified as S. iniae. The total DNA was extracted from liver and kidney tissues of diseased A. baerii for PCR detection of AciHV-2 polymerase gene, and a 501 bp target band was amplified. A phylogenetic tree based on the sequences indicated that the two samples(QL2, PZ2)and other AciHV-2 strains were clustered together in a branch. Histopathologically, degeneration, necrosis, hemorrhage and infiltration of the inflammation cells occurred in multiple tissues and organs, particularly obvious in liver, kidney, gill, spleen and intestine. A large number of cells invaded by herpesvirus-like particles with a diameter of 200-220 nm and streptococcus with a diameter of 0.7-0.8 microns were observed using electron microscopy, which caused cell damage. In conclusion, the etiology of the diseased A. baerii was a co-infection of S. iniae and AciHV-2.

Abstract:There was an outbreak of Bacterial Ulceration Syndrome (BUS) of Apostichopus japonicus in two culture ponds located in Dalian, Liaoning Province and Dongying, Shandong Province, in October 2019. Epidemiological investigation detected a kind of free-living turbellarians in the culture system including the body surface of A. japonicus, the water and the sediment. Morphological observation of this species revealed that the body length ranged from 0.96 to 3.26 mm and the body width range from 0.49 to 1.93 mm. The body color was yellow or yellowish-brown. Its head was blunt round shape, with a pair of dark red rodlike ocellus. Two caudal lappets lay coordinately on both sides of its tail.Microscopic examination revealed that symbiotic Zooxanthella within the epidermis and its body was covered with cilia. It was hermaphrodite, with two genital openings behind the mouth. The 18S rDNA gene sequence showed 99.64% identity with Heterochaerus australis. Based on the morphological and genetic analysis, it was identified as H. australis. The ecological study showed that this species was photophobic, its suitable temperature was 18-24 °C, its suitable pH was 5.5-8.0 and its suitable salinity was 20-40. This species also showed strong ability of polar regeneration. The co-cultivation experiment of this species and A. japonicus showed that H. australis can adsorb on the surface of sea cucumber, causing ulceration on the body wall and eventually the death of sea cucumber. However, there was no parasitic phenomenon in the body cavity, intestine and respiratory tree of the sea cucumber. All the results indicate that H. australis is a new enemy species for A. japonicus in pond culture.

Abstract:In July 2018, an outbreak of unknown disease occurred in the silver pomfret (Pampus argenteus) cultured in a farm in Xiangshan, Ningbo, with a cumulative mortality rate of over 80%. To investigate the cause of disease outbreak, histopathological analysis, transmission electron microscope observation and molecular biological detection and analysis were carried out to identify the pathogen, so as to help us to understand and control the disease.The clinical manifestations of sick P. argenteus are anorexia, body imbalance, swelling of the spleen, and abnormal color of the liver. Histopathological analysis showed the presence of basophilic enlarged cells with a diameter of 10~15 μm in diseased fish spleen, liver and kidney. Further transmission electron microscopic observation revealed the presence of inclusion bodies and a large number of virus particles (a diameter of 140-160 nm) in cells from spleen and kidney tissues. Moreover, results of the iridovirus-specific PCR test showed that all these tissue samples collected from diseased fish were positive for iridovirus. To further detect the presence of iridovirus, the major capsid protein (MCP) gene of virus in P. argenteus was amplified, cloned and sequenced. Sequence similarity analysis showed that silver pomfret-derived virus (SPDV) shared the highest homology with the large yellow croaker iridovirus (LYCIV) (GenBank accession number: AY779031.1) MCP, with a sequence similarity of 99.76%. In a phylogenetic tree, SPDV and LYCIV are closely grouped with red sea bream iridovirus (RSIV) strains. Thus, we concluded that the virus that infected silver pomfret belongs to the Iridovirus family, Megalocytivirus genus, RSIV group. In summary, this paper reported Megalocytivirus infection in cultured P. argenteus for the first time. This study will provide an important reference for the diagnosis and prevention of P. argenteus iridescent virus disease.

Abstract:To investigate the immune response of zebrafish to iron-related proteins and SWCNT-encapsulated proteins, we overexpressed and purified two iron-related recombinant proteins (A0KLQ6 and A0KH89) from Aeromonas hydrophila. We then vaccinated Danio rerio with these proteins and their SWCNT-encapsulated counterparts via both intraperitoneal injection and immersion immunization, and found they evoked a strong immune response in D. rerio. When challenged with virulent A. hydrophila, D. rerio administered 5 μg intraperitoneal injections of A0KHR9, A0KLQ6, A0KH89, SWCNTs-A0KHR9, SWCNTs-A0KLQ6 and SWCNTs-A0KH89 obtained high relative percent survivals (RPSs) (61.60%, 65.39%, 84.96%, 68.10%, 77.50% and 90.43%, respectively). Meanwhile, the D. rerio administered 40 mg/L immersion immunizations of aboved samples had 30.80%, 25.85%, 37.80%, 63.60%, 73.74% and 68.49% RPSs, respectively. These results indicate that the outer membrane proteins A0KLQ6 and A0KH89 can be used as potenial subunit vaccine candidates against A. hydrophila infection, and the SWCNTs-encapsulated counterparts can significantly improve the immune effect in both immunization ways. Our studies may provide a theoretical basis for the development of vaccines and adjuvants candidates

Abstract:Histones are central components of nucleosome and chromatin, which play critical roles in diversifying chromatin structure, transcriptional regulation, ontogenesis and so on. Although there were a lot of reports on the effects of piscine histones in the development, gene transcription regulation and anti-microbial properties of histone-derived antimicrobial peptides, the roles of histone nucleotide polymorphism in pathogen infection have not been reported in any species of vertebrates. In the present study, we found that nucleotide polymorphisms were abundant in zebrafish (Danio rerio) and grass carp (Ctenopharyngodon idella) H2A. There was 90%-100% identity among D. rerio and C. idella H2A variants at the nucleotide level. At most 3 sites of amino acid mutation existed between H2A variants from D. rerio or C. idella. The results from the in vitro and in vivo studies showed that the nucleotide polymorphism of D. rerio and C. idella H2A significantly affected the antibacterial activities of H2A. Furthermore, the overexpression of D. rerio antibacterial H2A variants in D. rerio embryos and/or larvae not only has the immune enhancement effect, but also enhances the resistence of D. rerio larvae against Edwardsiella piscicida infection. The present study established the immunological basis of histone H2A variants with nucleotide polymorphism in disease susceptibility or disease resistance.

Abstract:To clarify the biological and physicochemical properties of Anguillid herpesvirus (AngHV), an AngHV strain, NA16108, isolated from the diseased Anguilla anguilla of "mucus sloughing and hemorrhagic septicemia disease" was used to study its replication characteristics and infection sensitivity to some fish continuous cell lines, and its survivability to heat, acid, alkali, chloroform, ether and other physical and chemical factors was further analyzed. The results indicated that AngHV infected eel ovary cells (EO) showed typical sequential cytopathic effects (CPE), and many typical herpesvirus-like viral particles could be seen in the infected cells. AngHV could propagate stably in EO cells, and the suitable temperature for propagation is 25 °C to 27 °C, but it could not propagate in epithelioma papilloma cyprinid cells (EPC), grass carp ovary cells (CO), fathead minnow cells (FHM), chinook salmon embryo cells (CHSE-214), rainbow trout gonad cells (RTG-2), bluegill fry cells (BF-2). The titer of AngHV slightly decreased after 30 min treatment at 37 °C, and it could be completely inactivated after 30 min treatment at 56 °C. AngHV is sensitive to acid (pH 3.0), but resistant to alkali (pH 10.0), and highly sensitive to chloroform, ether and other organic solvents. These results can provide references for the prevention and control of AngHV diseases.

Abstract:Acipenser sinensis belongs to the cartilaginous fish, which is between the cartilaginous fishes and teleost fishes. It is an important branch point in the evolutionary tree of the biological immune system. It is a living fossil for the study of the evolution of fish and vertebrates, and plays an extremely important role in the evolutionary history of fish and vertebrates. In order to understand the immunomodulatory effect of A. sinensis IFN-γ, IFN- γ sequence was obtained by transcriptome of A. sinensis, the recombinant plasmid of pTRI-st-IFNg was constructed and transformed into the BL21 Escherichia coli for expression and expressed in competent prokaryotic cells. SDS-PAGE analysis showed that the size of IFN-γ recombinant protein was 19.17 ku, and the concentration was 0.998 mg/mL. Quantitative PCR showed that the antiviral genes Mx, Viperin, CXCL11-L1,CXCL11-L2 (CXC subfamily of chemokines) and antiviral protein Epithelioma papulosum cyprini cells (EPC) P,N,G were induced, which indicated that IFN-λ activated antiviral activity. Additionally, spring viremia of carp virus (SVCV) replication as well as the production of cytopathic effect (CPE) was significantly inhibited. Results of incubation with Aeromonas hydrophila, A. intermedia and A. veronii showed a significant inhibition on their growth and activity. In conclusion, this study provides partial theory for the understanding of IFN-γ bioactivity in A. sinensis.

Abstract:In order to obtain promising tools for improved diagnostics and new vaccine development, we purified Cyprinid herpesvirus 2 (CyHV-2) particles and identified more immunologic proteins in CyHV-2 particles by LC-MS. Firstly, the caudal fin of Carassius auratus gibelio (GiCF) cell line was infected with CyHV-2, and viral particles were purified from the cell supernatant by sucrose density gradient in combination with ultracentrifugation. The purified virions were observed by transmission electron microscope. Then the prepared purified virions were used to immunize mice to prepare an anti-CyHV-2 polyclonal antibody, and at the same time purified virions were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with coomassie brilliant blue, and then subjected to Western blotting analysis and mass spectrometry identification using anti-CyHV-2 polyclonal antibody. Transmission electron microscope (TEM) results showed that a large amount of intact CyHV-2 virus particles were detected at 50%-66% sucrose density and virus particles without capsule were occasionally observed. Western blotting results showed that some protein bands had specific immunoreactivity with anti-CyHV-2 polyclonal antibody. 8 major immunogenic proteins were further identified as ORF92, ORF115, ORF25, ORF57, ORF66, ORF72, ORF131and ORF132 through mass spectrometry identification. Among them, ORF92, ORF66 and ORF72 are capsid proteins, and ORF115, ORF25, ORF131 and ORF132 are membrane proteins. The capsid protein and membrane protein of the virus play an important role in virus infection. In conclusion, the major immunogenic proteins of CyHV-2 virion were identified. This was fundamental and applied research on CyHV-2. Indeed, this knowledge is crucial for understanding the biology and pathogenesis of CyHV-2 infection and for facilitating the development of efficacious protein-based diagnostic methods and vaccine candidates.

Abstract:To clone Type I interferon (IFN-1) from epithelioma papulosum cyprini cells (EPC) of Pimephales promelas, the ifn-1 gene was amplified from EPC by RT-PCR, and the recombinant plasmid of pET-32a-IFN-1 was constructed and transformed into the host strain Transetta for expression. Sequence analysis showed that the coding region of ifn-1 gene was 552 bp in length and encoded 184 amino acids, which was closely related to grass carp interferon 1(CiIFN1). Recombinant soluble IFN-1 (rIFN-1) was induced in Transetta by IPTG and obtained by Ni-affinity purification. Then, anti-rIFN-1 polyclonal antibody was prepared by immunizing New Zealand white rabbits with rIFN-1 and could be used to detect endogenous IFN-1 in EPC cells. Quantitative PCR showed that the antiviral protein Mx1 was induced and spring viremia of carp virus (SVCV) replication as well as the production of cytopathic effect (CPE) was significantly inhibited when EPC was pre-incubated with rIFN-1, which indicated that rIFN-1 possessed antiviral activity in EPC cells.

Abstract:Macrophage migration inhibitory factor (MIF) is evolutionarily ancient and has been found across kingdoms including animals, plants and bacteria. In mammals, MIF has been suggested as chemokine-like cytokine with enzymatic activities, which plays a critical role in inflammatory response against pathogen infections. In this study, we cloned a MIF-like (named after AjMIF) gene for the first time in Japanese eel, Anguilla japonica. The AjMIF precursor processes MIF signature sequence motif, Cys₅₇-Ala-Leu-Cys₆₀, for its thiol-protein oxidoreductase activity, and several conserved residues which are critical for its isomerase activity, such as Pro₂ and Cys₈₁. Expression analysis showed that AjMIF is expressed in various organs / tissues with the highest in liver, followed by middle kidney and intestine. The expression level of AjMIF was significantly increased in head kidney, middle kidney and swim bladder after challenged with lipopolysaccharides at 8 hours post-injection (hpi). Significant increase in AjMIF expression was also observed in gill, skin and intestine at 8 hpi following stimulation with polyinosinic-polycytidylic acid. In addition, significant increase of AjMIF mRNA in gill and intestine was observed at 8 hpi, and in gill at 16 and 24 hpi, and in skin at 24 hpi when infected with Edwardsiella tarda. Furthermore, pH-dependent tautomerase activity of AjMIF has also been found by a recombinant rAjMIF using a prokaryotic expression system. Our results showed that 1 nmol of rAjMIF exhibited 2.6 U of tautomerase activity at pH 6.2, but 36.6 U at pH 8.0. Overall, our study provides a basis for future research aiming at a better understanding the functions of MIF in fish immune system.

Abstract:Drug resistance mainly caused by the abuse of antibiotics is becoming more and more serious, therefore there is an urgent need to develop novel antibacterial agents. Due to its safety, non-resistance, and high stability, the antibacterial activity of silver ion(Ag+) has attracted the attention of more and more researchers. In order to explore the antibacterial mechanism of Ag+, several common aquatic bacteria, named (Escherichia coli, Aeromonas hydrophila, Streptococcus agalactiae, Bacillus subtilis, Edwardsiella ictaluri), were used to test the Ag⁺ tolerance or bactericidal activity and its "zombie effect" ( the bacteria killed by Ag⁺ can kill other fresh bacteria). The results of tolerance experiments of bacteria to Ag⁺ showed that Ag⁺ had significant growth inhibition on the all five bacteria. The higher concentration of Ag⁺, the more apparent "zombie effect" was observed between the same species of bacteria as well as different species of bacteria. To further investigate the mechanism underlying of the antibacterial activity of Ag⁺, the Ag⁺ treated A. hydrophila and S. agalactiae were observed by transmission electron microscopy (TEM). The TEM results demonstrated that Ag⁺ could cause the separation of the cytoplasmic membrane (CM) from the cell wall and discharge of cytoplasmic organelles, so as it could cause the lysis of the bacterial cell wall. In summary, this study reveals the prolonged antibacterial mechanism of Ag⁺ which will pave a new way for the development of novel antibacterial agents.

Abstract:In order to determine the cause of the large number of deaths of the sick American eel (Anguilla rostrate) in an aquaculture farm in Haikou (Hainan), one dominant strain (named AB01) was isolated from the liver and spleen of the diseased American eels. The dominant strain AB01 was identified as Acinetobacter baumannii based on the results from physiological and biochemical testing, sequence alignment of 16S rDNA gene and phylogenetic analysis. The challenge test in healthy American eels by artificial reentry indicated that A. baumannii was the pathogenic bacteria. Further analysis of pathogenicity showed that the semi-lethal concentration of A. baumannii (AB01) was 4.63×10⁶ CFU/mL. Histopathological observation of liver from diseased Anguilla anguilla showed the arrangement of some hepatic cords was disordered, and the cells were swollen, together with dissolved nucleus, loose cytoplasm, and fuzzy structure. Moreover, a lot of vacuoles of different sizes were present in liver. In addition, spleen from diseased Anguilla rostrata presented sparse cytoplasm and extensive injury. The results of antimicrobial susceptibility test showed that A. baumannii (AB01) was resistant to many antibiotics, but was highly sensitive to cephalothin, cefazolin, azithromycin, amikacin, enoxacin, rifampicin, ciprofloxacin, ofloxacin, and medium sensitivity to nine antibiotics, such as florfenicol and so on. Moreover, A. baumannii (AB01) was resistant to twenty antibiotics, such as aztreonam and so on. Thus, this study provides scientific support for the prevention and control of the disease of the American eel.

Abstract:Bacterial septicemia is a systemic inflammatory reaction mainly caused by the infection of Aeromonas hydrophila. Excessive development of inflammation may lead to septic shock or death in fish. A large number of studies have confirmed that miRNA is involved in the regulation of immune response after bacterial infection. To explore the regulatory mechanism of miR-462 in Ctenopharyngodon idella kidney (CIK) cells infected with A. hydrophila, the expression profiles of miR-462 upon A. hydrophila infection was detected by real-time quantitative PCR; the target genes of miR-462 were predicted by RNAhybrid software, and identified by dual-luciferase reporter assay system; in addition, the regulatory effect of miR-462 on downstream genes was analyzed. The results showed that the expression of miR-462 changed significantly after A. hydrophila infection, indicating that miR-462 participated in the regulation of immune response. Dual-luciferase reporter assay revealed that cx32.2, slc9a3.1 and tbk1 are the target genes of miR-462, which is further confirmed by the overexpression and inhibition experiments of miR-462. The expression of slc4a4a, tnfrs5, cxcl9 and cxcl11 were suppressed after miR-462 antagomir was transfected, which proved that miR-462 could affect the function of the downstream genes by targeting slc9a3.1 and tbk1. Our results may provide a theoretical basis for investigating the molecular mechanism of miR-462 regulating immune response in C. idella.

Abstract:Antimicrobial peptides (AMPs) are groups of small molecular polypeptides, which are important parts of host innate immune system, having perfect inhibiting effects or killing effects against Gram negative bacteria, Gram positive bacteria, virus and parasites. Due to their non-pollution, no residue, broad-spectrum antibacterial activity and no drug resistance, AMPs are expected to replace antibiotics in the prevention and control of pathogenic diseases in aquatic animals. In recent years, AMPs have been reported in aquatic animals including aquatic crustaceans, aquatic mollusks, fish and amphibians. However, the classifications and immune mechanisms of AMPs need further investigation. In this paper, the AMPs from different aquatic animals were classified, and their structural features and functions were analyzed. The AMPs of aquatic crustaceans were mainly divided into penaeidins, crustins and anti-lipopolysaccharide factors (ALFs), which possessed different effects against aquatic pathogens. The AMPs of aquatic mollusks include defensins, mytilins, myticins, mytimycins and big defensins, which have perfect inhibiting effects against bacteria and fungus. Fish possess complex AMPs types, including piscidins, β-defensins, hepcidins, cathelicidins, liver-expressed antimicrobial peptide 2, (LEAP-2) and NK-lysins. Different types AMPs in fish shared distinct structural features and immune functions. Amphibian AMPs include the β-defensins, cathelicidins, andricin 01 of Caudata, and aureins, brevinins, bvombinins, buforins, cathelicidins, esculentins, magainins and temporins of Anura. The immune mechanisms of AMPs including direct killing effects, non-membrane target effects and immune regulation were also analyzed. This paper would provide a basis for the future studies of AMPs and their applications in aquatic animals.

Abstract:To investigate the effects of the oral administration of exopolysaccharides from Lactococcus lactis Q-9 (EPS-9) on the immunoregulatory properties, antioxidant activities and resistance against Aeromonas hydrophila in Cyprinus carpio, the purified EPS-9 (250, 500 and 1 000 μg/mL) were co-cultured with the head kidney cells of C. carpio. The proliferation and phagocytosis activities of the head kidney cells, and the concentration of nitric oxide (NO) and cytokines in the culture medium were determined. Next, 300 C. carpio [(47.66±0.43) g)] were randomly divided into five groups; the two control groups (negative and positive) were administered sterile PBS and the three treatment groups were administered different concentrations of EPS-9 (250, 500 and 1 000 μg/mL) for seven days. Subsequently, the positive and treatment groups were infected with A. hydrophila, and the negative group was treated with sterile PBS for 24 h. The concentration of NO, cytokines, lysozyme (LZM) and alkaline phosphatase (AKP) in serum, the antioxidative indexes (T-AOC, T-SOD, CAT, GSH, GSH-Px, MDA) in the hepatopancreas of the C. carpio were tested. We observed that EPS-9 could significantly enhance the proliferation and phagocytosis activities, besides inducing the production of NO, pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and anti-inflammatory cytokines (IL-10, TGF-β) in vitro. The in vivo experimental results revealed that EPS-9 significantly enhanced NO production, protein levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), LZM and AKP activities in the serum, and the antioxidant level in the hepatopancreas compared to that observed in the negative group prior to A. hydrophila infection. NO, pro-inflammatory cytokines, LZM and AKP activities were significantly lower than that in the positive group after infection. However, whether infected or not, the expression of anti-inflammatory cytokines (IL-10, TGF-β) increased significantly in the EPS-9-treated groups. Therefore, the results suggested that EPS-9 could enhance the non-specific immunity, antioxidant activities and anti-A. hydrophila activity of C. carpio in vitro and in vivo, and could be used as a safe and effective feed additive in aquaculture.

Abstract:In order to investigate the relationships among the C-Myc expression, glutamine metabolism and replication of NNV. First of all, the C-Myc gene (GF-1-C-Myc) from Epinephelus coioides fin cell (grouper fin cells, GF-1) was cloned. The full length of GF-1-C-Myc gene cDNA was 814bp with 285bp ORF, encoding 95 amino acid (aa) with leucine zipper domain and helix-ring-helix (HLH) domain. GF-1-C-Myc protein was expressed and purified, and its polyclonal antibody was generated. The expressions of GF-1-C-Myc gene and the replication of NNV were monitored by real-time quantitative PCR (qRT-PCR) and immunoblotting (WB). The results showed that lack of glutamine could inhibit both the expression of GF-1-C-Myc gene and replication of NNV, while additional glutamine could promote both the expression of GF-1-C-Myc gene and replication of NNV. In addition, the expression of GF-1-C-Myc gene was up-regulated in GF-1 cells infected with NNV, and the glutamine in the medium was significantly consumed. Taken together, GF-1-C-Myc gene was involved in the regulation of glutamine metabolism in the cell, subsequently facilitated the replication of NNV. Our results will shed a new light on the prevention and control of NNV infection.

Abstract:In order to make a further study of the pathogenic mechanism of Acinetobacter pittii an emerging fish pathogen and provide technical reserves for the prevention and control of fish diseases caused by A. pittii, we selected A. pittii strain Ap-W20 for the whole genome sequencing and analysis of its virulence characteristics. The results showed that strain Ap-W20 contained a chromosome of 4 399 705 bp with an average G+C content of 38.78% and 4 230 coding sequences (CDS). Particularly, Ap-W20 carries four previously unreported plasmids. The GenBank accession numbers of Ap-W20 are CP027658–CP027662. The ANI analysis result confirmed that Ap-W20 belonged to A. pittii, and Ap-W20 had close similarities with A. pittii AP_882 from human (96.69%) and A. pittii PHEA-2 (96.55%) from environment. Comparative genomic analysis of Ap-W20 with AP_882 and PHEA-2 found that the numbers of gene islands, prophages and plasmids in Ap-W20 were the largest, which is likely related to their respective isolation environments and specific pathogenicity. In comparison with the VFDB database, 286 virulence genes were predicted in the entire genome of Ap-W20, mainly related to adhesion and anti-phagocytosis. Ap-W20 also has Type I, II and VI secretion systems, multiple sets of two-component regulatory system, and bacteriocin synthesis gene cluster, etc., which suggests that there may be complex pathogenic mechanisms in A. pittii derived from fish. This study demonstrates the complete genome sequence and the virulence characteristics of A. pittii from fish, which may provide a basis for the disease control caused by A. pittii.

Abstract:In October 2018, crucian carp (Carassius auratus) in a farm located in Zigong, Sichuan Province, developed a communicable disease with a mortality rate of 80%. In order to explore the cause of this disease, sick fish were subjected to necropsy, pathological observation, molecular biological examination and artificial infection test. The clinical manifestations of diseased fish included leaving the shoal of fish to swim alone; staying on the water surface; bodies blackened and body surface had haemorrhages. The necropsy showed haemorrhage, swelling and necrosis of organs such as gills, liver and kidney; swimming bladder had a lot of blutene chloaide. Histopathological changes showed that the respiratory epithelial cells of gill were swollen, necrosis and sloughting. The kidneys showed focal necrosis. The spleen tissue was extensively degenerated and necrotic, and the hematopoietic system collapsed. Vacuolar degeneration and necrosis occurred in the liver. Transmission electron microscopy showed that there were four different sizes of virus particles in the spleen and kidney, which were virus DNA core, empty viral capsid, solid capsid and enveloped mature virions. It can be seen that immature virions are present in the nucleus, and mature virions are present in the cytoplasm. Cyprinid herpesvirus Ⅱ (CyHV-2) completed nucleic acid replication and nucleocapsid assembly in the nucleus, and the envelope protein was obtained after transmembrane membrane. After filtering the diseased fish tissue homogenate, it was inoculated into EPC and FHM cell lines, and there was no cytopathic effect after 3 generations of blind transmission. The helicase gene fragment of CyHV-2 was amplified by PCR, and a positive band appeared. The artificial infection test was carried out by intraperitoneal injection of tissue homogenate. The test group showed the same clinical symptoms as the natural infection fish and the control group was normal. The cumulative mortality rate of the test group was 80%. Phylogenetic analysis of helicase gene by the neighbor-joining method revealed that the homology with CyHV-2 (YZ-01) was 99%. The results demonstrated that CyHV-2 contributed to this disease outbreak.