Abstract:Streptococcus agalactiae is one of the major pathogens of tilapia (Oreochromis niloticus). At present, the virulence of strains was evaluated mainly by comparing the median lethal dose (LD₅₀) to tilapia. However, tilapia as experimental animals for evaluation of the virulence of S. agalactiae was often unstable. In this experiment, BALB/c mice were used as experimental animals, in order to establish a method for the determination of virulence of S. agalactiae. BALB/c mice were injected intraperitoneally with tilapia Streptococcus agalactiae to establish an infection model. The LD₅₀ of Streptococcus agalactiae to tilapia and mice were tested 3 times and the virulence of different Streptococcus agalactiae to tilapia and mice was determined. Results show that:by intraperitoneal injection, S. agalactiae can cause the death of mice within 24 hours. S. agalactiae could damage brain, liver, spleen, kidney and other tissues in mice. The LD₅₀ of S. agalactiae to tilapia and mice 3 times was 7.7×10⁷, 2.2×10⁸, 3.5×10⁹ CFU and 405, 361, 419 CFU, respectively. When S. agalactiae TFJ0901 and THN0901 infected tilapia (1.0×10⁷ CFU) and mice (100 CFU), the survival rates of tilapia and mouse were 100.0%, 6.7%±5.8%, and 100.0%, 0%, respectively, both of which were significantly (P<0.05) different. When S. agalactiae TFJ0901 and TFJ-F infected tilapia (3.0×10⁸ CFU) and mice (2 500 CFU), the survival rates of tilapia were 73.3%±11.5% and 80.0%±10.0%, respectively, which were not significantly (P > 0.05) different from each other, while the survival rates of mice were 13.3%±11.5% and 100.0%, respectively, which were of significant (P<0.05) difference. Taken together, BALB/c mice were successfully established as a stable model for virulence determination of S. agalactiae in tilapia. The determination of the virulence of different S. agalactiae in mice is consistent with tilapia, and this model was able to distinguish S. agalactiae with similar virulence that was difficult to be distinguished by using tilapia model.

Abstract:In order to investigate the difference of the intestinal community structure and its relationship with environmental factors in response to disease, the intestines of healthy and diseased Trachinotus ovatus were used to analyze the bacterial community composition and diversity as well as the culture waters and pellet feed. The five samples were studied by using Illumina HiSeq high-throughput sequencing and biological information analysis method. Compared with the intestinal microflora of the healthy pompano, the relative abundances of Spirochaetes significantly increased in the diseased pompano, whereas, the Firmicutes and Bacteroidetes exhibited an opposite pattern. The bacteria species in diseased pompano intestine only accounted for 54.94% in healthy pompano. There was 73.46% of the OTUs in the healthy ovate intestine, the same as that in the culture waters, and 70.58% in the feed, while the percentage dropped to 17.98% and 38.95% in the diseased pompano. Notably, the disease pompano had a higher relative abundance of Vibrio ponticus than healthy pompano being 78.90% and 17.19%, respectively. In addition, the relative abundance of Photobacterium leiognathi in healthy pompano was 54.53%, but absent in diseased pompano. The intestinal bacterial composition was relatively stable but there were still some difference between healthy and diseased pompano. After the occurrence of disease, the bacterial diversity declined markedly. The culture water and feed had a close correlation with the healthy pompano intestine but low influence on diseased pompano in bacterial species composition.

Abstract:The "white spot disease" caused by the parasitic infection of Cryptocaryon irritans has become one of the most serious diseases in the production of Larimichthys crocea. This study aims to investigate the pathogenicity of C. irritans to L. crocea, as well as the effect of Chinese herbal compound HD-2 on inhibiting the generation reproduction of parasite C. irritans. The number, size, the hatching rate and number of hatched theronts of tomonts produced by natural sick L. crocea were investigated. Then the survival time and infectivity of theront was determined in vitro. The effects of Chinese herbal compound HD-2 on theront, trophont, and tomont, and the therapeutic effects of oral HD-2 on diseased L. crocea were studied. The results showed that the total number of tomonts produced by L. crocea was positively correlated to the fish body weight; the average diameter of tomonts were (340.8±64.9) μm and the hatching rate was 76.4%, and each tomont could produce (280±42.5) theronts; the theront could survive for more than 24 h in vitro and lost their infectivity in 20 h after hatching. The 90% lethal dose (LD₉₀) of L. crocea infected with C. irritans in 7 days was 9 712 theronts/fish and the median lethal dose for 50% (LD₅₀) was 4 366 theronts/fish. HD-2 could kill theront effectively. After oral administration with different doses of HD-2 (control, 0; G1, 5 g/kg; G2, 10 g/kg; G3, 15 g/kg) for 30 days, the relative infection intensity of the L. crocea groups challenged with LD₅₀ decreased by 32.0%, 44.5% and 51.8%, respectively. Meanwhile, the volume of tomonts reduced by 35.4%, 36.1% and 37.3% and the hatching rate reduced by 16.3%, 23.3% and 27.9%, respectively. The fish survival rate of G1, G2 and G3 challenged with LD₉₀ was 40.0%, 55.0% and 58.3%, which was significantly higher than the control group (only 8.3%). Using HD-2 oral treatment for 15 d, the tomonts produced from diseased fish reduced 73.1%, 87.7% and 93.8%, and the fish mortality rate of the three groups were 58.3%, 36.7% and 21.7%, which were significantly lower than the control group (83.3%). The physiological status of L. crocea in the treated group was significantly better than that of the control group. Therefore, HD-2 oral treatment could effectively interrupt the secondary infection of C. irritans, suppress the formation and development of tomonts, reduce the tomont hatching rate and the number of hatching theronts, and then inhibit the generation reproduction of parasite C. irritans.

Abstract:The antibacterial effect of the drugs against Vibrio alginolyticus in vitro and the drugs on the resistance to diseases of Epinephelus coioides were studied. This study aimed to screen traditional Chinese medicines formulas and Chinese and western medicines formulas which can prevent V. alginolyticus diseases of E. coioides. We had the bacteriostatic test on traditional Chinese medicines formulas against V. alginolyticus in vitro. The ratios of Terminalia chebula, Radix paeoniae and Glycyrrhiza uralensis of Chinese medicines formulas Ⅰ, Ⅱ, Ⅲ were 0.9:1.3:0.9, 1.2:0.9:1.0, and 1:1:1, respectively. The groups with 3 replicates per treatment of feed containing the drugs feed additives at 2.2% respectively, were fed to E. coioides. The ratio of T. chebula, R. paeoniae and G. uralensis were different in Chinese medicines formulas trial groupⅠ, Ⅱ, Ⅲ. The ratios of T. chebula, R. paeoniae, G. uralensis and enrofloxaci were different in Chinese and western medicines formulas trial groupⅣ, Ⅴ. Results revealed that the concentration of Chinese medicines formulas Ⅲ (200 mg/mL) was the best formula. The mortality for 40 d in test groups were as follows:control groupⅡ > control group Ⅳ > control group Ⅲ > trial group Ⅲ > trial groupⅡ > trial groupⅠ > trial group Ⅴ > trial group Ⅵ > trial group Ⅳ > control groupⅠ. The ratio of T. chebula, R. paeoniae and G. uralensis of Chinese medicines formulaⅠwas 0.9:1.3:0.9. The ratios of T. chebula, R. paeoniae, G. uralensis and enrofloxaci in Chinese and western medicines formula Ⅳ were different. These are the best formulas. The Chinese and western medicines formula is better than the Chinese medicines formula. Results showed that by adding the formulas, the capacity of disease resistance can be improved.

Abstract:To compare the differences in gut microbiota and antibiotic resistance genes (ARGs) between Litopenaeus vannamei and Macrobrachium rosenbergii, microbiota community structure and microbiota diversity of two shrimps were analyzed by high-throughput sequencing and denaturing gradient gel electrophoresis (DGGE), and 38 kinds of ARGs in the gut bacteria of two shrimps were detected. The effective bacterial sequences of L. vannamei and M. rosenbergii were 42 795 and 40 713. The results showed that the numbers of operational taxonomic units (OTUs) were 124 and 82, and the bacterial types with clear classification status were 5 phyla, 17 genera and 5 phyla, 16 genera. The dominant group in the gut bacteria of L. vannamei is Proteobacteria, which accounts for 75.45%. The dominant genera are Paracoccus (25.83%) and Acinetobacter (25.24%). The dominant group in gut bacteria of M. rosenbergii is Firmicutes (49.74%), and the dominant genera are Lactococcus (49.01%) and Vibrio (29.98%). The Shannon index of the gut bacteria (2.19) of L. vannamei was higher than that of M. rosenbergii (1.78), indicating that the gut bacteria diversity of the former was greater than that of the latter. The analysis results of DGGE fingerprint were consistent with those of high-throughput sequencing, and the bacterial diversity of the two shrimps was significantly different. The PCR results showed that the gut bacteria of L. vannamei carried 15 ARGs and gut bacteria of M. rosenbergii carried 14 ARGs. Our study revealed that the community diversity, OTU richness, total number of species and ARGs in the gut bacteria of L. vannamei were higher than those of M. rosenbergii, which provided a theoretical basis for the subsequent excavation of gut microbial resources.

Abstract:In order to investigate the pathogen of the diseased tongue sole Cynoglossus semilaevis, three strains of Gram-negative bacteria isolated from diseased C. semilaevis were tentatively named strains ST-1, ST-3 and ST-6, which were identified by physiological and biochemical characteristics, antimicrobial susceptibility test, 16S rDNA gene sequences analysis and histopathology examination. Furthermore, the SYBR Green Ⅰ quantitative real-time PCR (qRT-PCR) targeting the outer membrance protein (OMP) gene was established. The biochemical results of the isolate ST-1, ST-3 and ST-6 were consistent with the properties of V. vulnificus. The BLAST alignments of the 16S rDNA gene sequence showed that the isolates ST-1, ST-3 and ST-6 shared the identities above 99% with type strain Vibrio vulnificus ATCC 29307 (X74727). The phylogenetic tree indicated the isolates clustered with V. vulnificus ATCC 29307. The isolates were eventually identified as V. vulnificus. Antimicrobial susceptibility testing showed that the isolates were highly susceptible to cefixime, cefoperazone, streptomycin, imipenem and florfenicol. The challenge experiments showed that the isolated strains were pathogenic on C. semilaevis and zebrafish(Danio rerio), and similar symptoms were observed with the natural infection. Histopathology examination revealed the hyperplasia of the epithelial cells in the primary lamella, the swelling of the secondary lamella and the increase of the number of granulosa cells and mucus cells, hepatic sinusoid and the congestion of central vein in the liver, hemorrhagic-necrotic lesion in the kidney, the atrophy of the lamina propria and the increase of the number of mucus cells in the intestine. The SYBR Green I quantitative PCR (qPCR) method based on the OMP gene was further established and the results showed that the correlation coefficient of standard curve was 0.995 and the lowest limit of detection was 1.88×10² CFU/mL. The results of this study will lay a foundation for the pathogenesis and prevention of C. semilaevis vibriosis, which can provide scientific reference for the molecular diagnosis of the disease.

Abstract:Carassius auratus herpesvirus (CaHV) is a pathogen that can cause crucian carp disease with acute gill hemorrhages and high mortality. Interactions of functional genes with cellular components, such as cellular organs, are needed by viruses to complete infection and replication. In the present study, bioinformatic analysis, PCR amplification, gene cloning, constructing recombinant plasmids, and fluorescence observation of the fish cells (epithelioma papulosum Cyprinid, EPC) cotransfected with cellular organs tagged plasmids were used to analyze the characteristics, subcellular localization and colocalization with cellular organs of the protein encoded by the gene CaHV ORF31R (CaHV-31R). As the results show, the encoding protein CaHV-31R was composed of 313 amino acids, which consists of a transmembrane domain (248-270 aa) and a typical domain of RNase E/G protein family (40-182 aa). The multiple alignment with homology proteins from 5 different fish hepesviruses exhibited higher identity (100% and 80.7%) with the Janpanese strain ST-J1 and Chinese strain SY-C1, which were both the members of type Ⅱ Cyprinid hepesviruses, centered identity (26.5%) with CyHV-1 which was the representative member of type I Cyprinid hepesviruses, and the lowest identity (20.7% and 18.2%) with the Germanic strain CyHV-3(also named Koi herpesvirus, KHV) and Chinese strain (CyHV-3-GZ11), which were both the members of type Ⅲ Cyprinid hepesviruses. The plasmids pEGFP-31R was constructed by PCR amplification, gene cloning, and then used to transfect EPC cells. pEGFP-31R dispersed distribution in cytoplasm and a small part of spotty distribution were found circling around outside the nucleus. CaHV-31R was used to colocalize with two celluar organs with monolayer membrane in cytoplasm, which was endoplasmic reticulum and Golgi apparatus. The results indicated that CaHV-31R was a gene of Carassius auratus herpesvirus, and was able to encode a protein that colocalizes with cellular organs.

Abstract:This paper studied the identification, drug resistance and the molecular resistance mechanism of five Vibrio strains isolated from diseased Cynoglossus semilaevis. The five strains were identified as V. cyclotrophicus by a biochemical test combined with a molecular method (16S rRNA, HSP60 and mreB genes). The broth dilution method was used to determine the antimicrobial susceptibility of the strains to 14 antimicrobials. The PCR amplification and DNA sequencing were used to detect antimicrobial resistance genes, integrons and the gene cassettes. The results indicated that the five strains were resistant to ciprofloxacin, streptomycin, erythromycin, florfenicol, ampicillin, SMZ, TMP/SMZ and rifampicin, and carried qnr VC, bla_CARB-17, strA, strB, sul genes. Class I intergron, qacEΔ1 and sul1 genes were present in JS291 and JS295 strains. The integron-gene cassettes of JS291 were encoding dihydrofolatereductase (dfr16), aminoglycoside-(3″)(9)-adenylyltransferase (aadA2), and the cassettes of JS295 were encoding rifampin ADP-ribosylating transferase (arr-3), dihydrofolate reductase (dfrA27), respectively. These findings suggested that the five strains exhibited multi-drug resistance and the multi-drug resistance phenotype may be closely related to the antimicrobial resistance genes.

Abstract:Fish skin mucus serves as the first line of defense against pathogens and external stressors. The mudskipper Boleophthalmus pectinirostris inhabit intertidal mudflats containing abundant and diverse microbial population, thus, the skin together with the mucus of B. pectinirostris are very important for the immune defense, osmotic pressure maintenance and adaptation to amphibian life. For exploring the proteomic profile of the skin mucus and understanding the molecular mechanism of B. pectinirostris adaptation to amphibious environments, the antibacterial activity was determined by agar diffusion plate method for the mucus and the serum samples. In addition, a growth curve inhibition method was used to compare the antibacterial activities of B. pectinirostris mucus before and after vibrio induction. Furthermore, the proteomic profile of natural B. pectinirostris mucus was identified by Shotgun mass spectrometry technology combining with skin transcriptome searching. The interaction network analysis of the identified proteins from mucus was performed by String software. Skin mucus was collected from B. pectinirostris after electrical stimulation. The mucus of B. pectinirostris showed a broad spectrum of antimicrobial activity more than serum of the same species, indicating that the mucus has a stronger antimicrobial activity than the serum. After vibrio induction, the antibacterial activity of mucus was slightly stronger for some gram-negative bacteria than that of un-induced mucus. A total of 97 proteins were identified from natural mucus of B. pectinirostris with a similar results from studies of other fish species, including actins, keratins, apolipoproteins, transferrins, calmodulins, ubiquitins, pentraxins, and various enzymes. However, some proteins, such as ubiquitin-like proteins and thymosin, were identified first from fish mucus. The identified proteins can be clustered into structural proteins, enzymes, material transport related proteins, immune proteins, and other proteins. Most of these proteins are known to be involved in immune and/or stress responses. Protein interaction analysis showed strong interactions among the identified proteins, such as actin, myosin, cofilin, filamin, apolipoprotein, transferrin, calmodulin, and superoxide dismutase. The proteomic profile established in this study could not only provide knowledge on the routes involved in mucosal innate immunity, but also put forward a non-invasive technique based on locating immune markers with a potential use for prevention and/or diagnosis of fish diseases.

Abstract:To isolate and identify lytic bacteriophages against AHPND-associated Vibrio parahaemolyticus and analyze their biological properties, lytic bacteriophages were isolated from Fujian and Mexico seawater samples by double-layer agar culture method, and one strain of V. parahaemolyticus strain associated with AHPND was used as the host bacterial strain for phage isolation and amplification. The phages were classified and identified by plaque shapes, restriction endonucleases, whole genome sequence, phylogenetic tree building and transmission electron microscopic technique. The lysis range and biological properties including optimal multiplicity of infection (MOI), thermal stability, pH stability, stability to peracetic acid and survival stability at different temperature were determined. The results showed that 3 lytic bacteriophages against AHPND-related V. parahaemolyticus were isolated, named P46, P48 and VP7. Their plaques were translucent circle. The plaque diameter of P46 was 4-5 mm. The plaque diameter of P48 and VP7 were 5-6 mm. The nucleotides of 3 bacteriophages were all dsDNA. P46, P48 and VP7 all had an icosahedral head with about 60 nm diameter. Their tails were about 18-20 nm in length and 8 nm in width. 3 phages belonged to the familiy Podoviridae. The cocktail of P46, P48 and VP7 lysed 91.5% of AHPND-associated V. parahaemolyticus strains and non-VP_AHPND 92.3% respectively. And they all could not identify non-V. parahaemolyticus bacteria. The optimal MOI of P46, P48 and VP7 were 0.001, 0.1 and 0.001 respectively. The optimal pH of P46 and VP7 were 6-8 and P48`s was 6-9. Three bacteriophages were stable at 60℃ and had higher survival rate at 4℃ for 50 weeks. P46, P48 and VP7 also tolerated peracetic acid with universal bactericidal concentration. Alignment with the sequences of the conserved protein sequences of NCBI showed that P46, P48 and VP7 had low homology with other bacteriophages. Bacteriophages P46, P48 and VP7 might be 3 novel Podoviridae bacteriophages. This study is the first report of lytic Podoviridae bacteriophages against AHPND-associated V. parahaemolyticus in China. The results may enrich the resources of bacteriophages against AHPND-associated V. parahaemolyticus and provide potential application of phages as a biocontrol agent.

Abstract:In order to study the effects of miRNAs in stripped snakehead (SSN-1) cell line upon snakehead vesiculovirus (SHVV) infection, SHVV-infected and mock-infected SSN-1 cells were analyzed by miRNA high-throughput sequencing. The expression levels of twenty-two miRNAs with levels equal to or higher than 1 000 reads per million (TPM ≥ 1 000) were determined by qRT-PCR with U6, β-actin, and 5S rRNA as internal control. When U6 gene was used as internal control, no significant changes were observed for β-actin upon SHVV infection, while 5S rRNA was upregulated in SSN-1 cells upon SHVV infection. The results showed that U6 gene or β-actin could be used as internal control genes for miRNAs in SSN-1 cells. Furthermore, fourteen miRNAs were selected and their effects on SHVV replication were analyzed. The results showed that eleven miRNAs (miR-27a-3p, miR-26a-5p, miR-30e-3p, etc.) promoted, whereas miR-150 and miR-216b suppressed, SHVV replication. Next, we found that high-expression miR-100-5p was upregulated at early stage of SHVV infection, while downregulated at later stage. Overexpression of miR-100-5p exhibited significant suppression of SHVV replication, while inhibition of expression of miR-100-5p promoted SHVV replication. Our findings presented valuable information for the study of molecular drugs against SHVV.

Abstract:The current study aimed to solve the problems of anaesthesia in clinical veterinary medicine of turtles. According to the results of pre-experiment, we selected the animal medicine recommended clinical anaesthetic drugs Telazol(tiletamine:zolazepam=1:1). We compared the anaesthetic effects of different dosages of Zoletil on Chinemys reevesiis, and the concentrations of zolazepam and tiletamine in plasma were detected by High Performance Liquid Chromatogram (HPLC), and analyzed with software PKsolver 2.0 to research the features of pharmacokinetics. Zoletil at a rate of 25 mg/kg was administered intramuscularly to C. reevesiis.The induction time, anaesthesia time and recovery time were 0.42, 4.33 and 4.14 h respectively. Zoletil on C. reevesiis anaesthesia was better, in line with the requirements of clinical surgery. The concentration-time curves of zolazepam and tiletamine in plasma all were conformed to a two-compartment open model. And the concentration-time equations of zolazepam and tiletamine were C=13.462e^-0.223t+2.127e^-0.055t, C=10.619e^-0.203t+0.279e^-0.047t, respectively. The major pharmacokinetic parameters were as follows:C_max=11.695 μg/mL, T_max=1.060 h and AUC=92.470 μg/(mL·h) in zolazepam; C_max=9.654 μg/mL, T_max=0.482 h and AUC=56.348 μg/(mL·h) in tiletamine. It suggested that blood concentration of zolazepam and tiletamine all reached 8.12 μg/mL and 8.96 μg/mL, respectively, which could cause healthy C. reevesiis into anaesthesia. To maintain anaesthesia, the blood concentrations of zolazepam and tiletamine should not be less than 7.18 μg/mL and 4.73 μg/mL.

Abstract:Hippocampus spp. is unique marine fish with special body morphology and life history traits. Compared with other marine fishes, the most special part of Hippocampus spp. is male breeding and strict monogamy. In addition, its unique body morphology, including curvaceous torso and horse-shaped head, has given them higher ornamental value. The trade volume of Hippocampus spp. is huge in the world, and the majority of the trade in Hippocampus spp. is for traditional Chinese medicine. In recent years, the destruction of wild habitats and overfishing lead to a sharp decline of the wild populations. Aquaculture of Hippocampus spp. has been considered as an effective way to solve the problem of decline of the wild populations and the unsustainable trade for traditional Chinese medicine. Therefore, the development of the Hippocampus spp. aquaculture is essential. However, the threat of various diseases has seriously affected the healthy development of the aquaculture industry. Since 2017, in the breeding process of the H. erectus in northern China, epidermis ulcer syndrome has frequently occurred, the morbidity is rapid and the mortalily is high. Epidermis ulcer syndrome has become a high incidence disease in the aquaculture process. Diseased H. erectus showed obvious symptoms, such as large rot area in body surface, darker and lackluster of the gills and liver, diseased H. erectus would die within 3 days. If the diseased H. erectus were not removed in time, a large number of seahorses would be infected. Although most fish diseases can be treated with antibiotics, the overuse of antibiotics may produce many potential threats, which are not conducive to the sustainable and healthy development of the aquaculture industry. Therefore, it is essential to further study the pathogens of various fish diseases and then we can develop biological control on this basis. Accordingly, the priority of research should be disease treatment and health management, especially disease treatment. Specific vaccines can be developed from isolated pathogenic bacteria, which provide effective way for disease treatment. In this study, dominant strains were isolated from the lesion tissue and intestine of H. erectus with epidermis ulcer syndrome. In order to determine the pathogenic bacteria, we carried out artificial infection, the infection results suggested that strain HDM-2 was the pathogen causing epidermis ulcer syndrome, and the LD₅₀ of strain HDM-2 was about 2.89×10⁸ CFU/mL. We identified strain HDM-2 with physiological and biochemical identification and molecular biological identification methods, and we observed strain HDM-2 under transmission electron microscope. 16S rDNA gene sequence analysis by BLAST in GenBank suggested that the similarity between strain HDM-2 and V. harveyi reached 100%. The observation results showed that HDM-2 was about 2.2 μm×4.75 μm, with peritrichous flagella and capsule. In order to study antibiotic susceptibility of strain HDM-2, we implemented the antibiotic sensitivity test by using Kirby-Bauerdiffusion method, and results showed that strain HDM-2 was highly sensitive to florfenicol. In order to study the pathogenicity of the pathogen to the host, we carried out histopathological observation of the diseased H. erectus. Observation results suggested that skin, liver and gills were severely damaged by HDM-2. This study would lay a foundation for the prevention and further study of the epidermis ulcer syndrome of H. erectus.