Nocardia seriolae mediates liver granulomatous chronic inflammation in Micropterus salmoides through pyroptosis.

Granulomatous diseases caused by Nocardia seriously endanger the health of cultured fish. These bacteria are widely distributed, but prevention and treatment methods are very limited. Chronic granulomatous inflammation is an important pathological feature of Nocardia infection. However, the molecular mechanisms of granuloma formation and chronic inflammation are still unclear. Constructing a granuloma infection model of Nocardia is the key to exploring the pathogenesis of the disease. In this study, we established a granuloma model in the liver of largemouth bass (Micropterus salmoides) and assessed the infection process of Nocardia seriolae at different concentrations by analysing relevant pathological features. By measuring the expression of pro-inflammatory cytokines, transcription factors and a pyroptosis-related protein, we revealed the close relationship between pyroptosis and chronic inflammation of granulomas. We further analysed the immunofluorescence results and the expression of pyroptosis-related protein of macrophage infected by N. seriolae and found that N. seriolae infection induced macrophage pyroptosis in vitro. These results were proved by flow cytometry analysis of infection experiment in vivo. Our results indicated that the pyroptosis effect may be the key to inducing chronic inflammation in the fish liver and further mediating granuloma formation. In this study, we explored the molecular mechanism underlying chronic inflammation of granulomas and developed research ideas for understanding the occurrence and development of granulomatous diseases in fish.

A Nanobody-Mediated Virus-Targeting Drug Delivery Platform for the Central Nervous System Viral Disease Therapy.

Viral diseases of the central nervous system (CNS) represent a major global health concern. Difficulties in treating these diseases are caused mainly by the biological tissues and barriers, which hinder the transport of drugs into the CNS. To counter this, a nanobody-mediated virus-targeting drug delivery platform (SWCNTs-P-A-Nb) is constructed for CNS viral disease therapy. Viral encephalopathy and retinopathy (VER), caused by nervous necrosis virus (NNV), is employed as a disease model. SWCNTs-P-A-Nb is successfully constructed by employing single-walled carbon nanotubes, amantadine, and NNV-specific nanobody (NNV-Nb) as the nanocarrier, anti-NNV drug, and targeting ligand, respectively. Results showed that SWCNTs-P-A-Nb has a good NNV-targeting ability in vitro and in vivo, improving the specific distribution of amantadine in NNV-infected sites under the guidance of NNV-Nb. SWCNTs-P-F-A-Nb can pass through the muscle and gill and be excreted by the kidney. SWCNTs-P-A-Nb can transport amantadine in a fast manner and prolong the action time, improving the anti-NNV activity of amantadine. Results so far have indicated that the nanobody-mediated NNV-targeting drug delivery platform is an effective method for VER therapy, providing new ideas and technologies for control of the CNS viral diseases. IMPORTANCE CNS viral diseases have resulted in many deadly epidemics throughout history and continue to pose one of the greatest threats to public health. Drug therapy remains challenging due to the complex structure and relative impermeability of the biological tissues and barriers. Therefore, development in the intelligent drug delivery platform is highly desired for CNS viral disease therapy. In the study, a nanobody-mediated virus-targeting drug delivery platform is constructed to explore the potential application of targeted therapy in CNS viral diseases. Our findings hold great promise for the application of targeted drug delivery in CNS viral disease therapy.

[Evaluation and Sources of Heavy Metal Pollution in the Surface Soil of the Qaidam Basin].

To understand surface soil heavy metal pollution characteristics, and the spatial distribution and sources of pollution in the main sedimentary features of the Qaidam Basin, a total of 129 topsoil samples(0-10 cm) were collected within a 25 km radius. The concentrations of As, Ba, Cr, Mn, Nb, Ni, Pb, Ti, Zn, Zr, and ten kinds of heavy metals were determined, the degree of contamination and potential sources quantitatively analyzed based on enrichment factors(EFs), the ground accumulation index(Igeo), and the absolute principal component-multiple linear regression(APCS-MLR) receptor model. The results showed that the surface soils of the Qaidam Basin have experienced pollution from ten kinds of heavy metals since the 1960s, with varying degrees of enrichment. As and Pb represent probable point source pollutants, and the basin as a whole shows negligible to low levels of pollution. The APCS-MLR analysis showed that heavy metal pollution derives from two sources, natural factors and anthropogenic(transportation) sources. Specifically, As, Cr, Mn, Nb, Ni, Ti, Zn, and Zr are associated with natural sources, while Ba and Pb are associated with both natural and anthropogenic sources.

Single-walled carbon nanotubes enhance the immune protective effect of a bath subunit vaccine for pearl gentian grouper against Iridovirus of Taiwan.

Iridovirus of Taiwan (TGIV) has been threatening the grouper farming since 1997, effective prophylaxis method is urgently needed. Subunit vaccine was proved to be useful to against the virus. Bath is the simplest method of vaccination and easy to be administrated without any stress to fish. In this research, we constructed a prokaryotic expression vector of TGIV's major capsid protein (MCP) to acquire the vaccine. Single-walled carbon nanotubes (SWCNTs) were used as the carrier to enhance the protective effect of bath vaccination for juvenile pearl gentian grouper (bath with concentrations of 5, 10, 20 mg/L for 6 h). Virus challenge was done after 28 days. Survival rates were calculated after 14 days. The level of antibody, activities of related enzymes in serums and expression of immune-related genes in kidneys and spleens were test. The results showed that vaccine with SWCNTs as carrier induced a higher level of antibody than that without. In addition, the activities of related enzymes (acid phosphatase, alkaline phosphatase, superoxide dismutase) and the expression of immune-related genes (Mx1, IgM, TNFαF, Lysozyme, CC chemokine 1, IL1-ß, IL-8) had a significantly increase. What's more, higher survival rates (42.10%, 77.77%, 89.47%) were provided by vaccine with SWCNTs than vaccine without SWCNTs (29.41%, 38.09%, 43.75%). This study suggests that the protective effect of vaccine that against TGIV with the method of bath vaccination could be enhanced by SWCNTs and SWCNTs could be a potential carrier for other subunit vaccines.

Pathological and immunological analyses of Thelohanellus kitauei (Myxozoa:Myxosporea) infection in the scattered mirror carp, Cyprinus carpio.

Thelohanellus kitauei is a spore-forming myxosporean parasite prevalent in scattered mirror carp (Cyprinus carpio) that generates numerous cysts in the intestine and causes mass mortality in fish. To investigate the infection and mortality induced by T. kitauei in pond-reared farms in Luo-Jiang (104°51'N, 31°31'E), southwest China, morphological and molecular analyses of infected fish were conducted. Natural and specific immune indicators were further evaluated to determine the immunological effects of response to parasitic infection. The infectious parasite was identified as Thelohanellus kitauei based on morphological, 18S rDNA and infectious characteristics. Scattered mirror carp was determined as the specific intermediate host of the parasite. However, T. kitauei still caused considerable damage to the fish, in particular, injury and blockage of the intestines, resulting in malnutrition and even death. The mature spores of T. kitauei colonize the intestinal submucosa of carp and form cysts of various sizes that block the intestinal tract and release spores into the enteric cavity upon rupture, leading to the next phase of T. kitauei growth. Moreover, T. kitauei-infected carp showed weaker innate immunity. IgM is involved in the fight against parasitic infection while cytokines, such as IL-6, IL-1ß and TNF-α, had an impact on infection processes. To our knowledge, this is the first report to show that T. kitauei infects and causes death in scattered mirror carp. Our collective findings from systematic pathology, morphology and immunology experiments provide a foundation for further research on infections by this type of parasite and development of effective treatment strategies.

A Yersinia ruckeri TIR Domain-Containing Protein (STIR-2) Mediates Immune Evasion by Targeting the MyD88 Adaptor.

TIR domain-containing proteins are essential for bacterial pathogens to subvert host defenses. This study describes a fish pathogen, Yersinia ruckeri SC09 strain, with a novel TIR domain-containing protein (STIR-2) that affects Toll-like receptor (TLR) function. STIR-2 was identified in Y. ruckeri by bioinformatics analysis. The toxic effects of this gene on fish were determined by in vivo challenge experiments in knockout mutants and complement mutants of the stir-2 gene. In vitro, STIR-2 downregulated the expression and secretion of IL-6, IL-1ß, and TNF-α. Furthermore, the results of NF-κB-dependent luciferase reporter system, co-immunoprecipitation, GST pull-down assays, and yeast two-hybrid assay indicated that STIR-2 inhibited the TLR signaling pathway by interacting with myeloid differentiation factor 88 (MyD88). In addition, STIR-2 promoted the intracellular survival of pathogenic Yersinia ruckeri SC09 strain by binding to the TIR adaptor protein MyD88 and inhibiting the pre-inflammatory signal of immune cells. These results showed that STIR-2 increased virulence in Y. ruckeri and suppressed the innate immune response by inhibiting TLR and MyD88-mediated signaling, serving as a novel strategy for innate immune evasion.

Multidrug-Resistant Aeromonas veronii Recovered from Channel Catfish (Ictalurus punctatus) in China: Prevalence and Mechanisms of Fluoroquinolone Resistance.

To emphasize the importance of the appropriate use of antibiotics in aquaculture systems, the prevalence of resistance to 25 antimicrobials was investigated in 42 Aeromonas veronii strains isolated from farm-raised channel catfish in China in 2006-2012. All experiments were based on minimal inhibitory concentrations (MICs), and susceptibility was assessed according to the Clinical and Laboratory Standards Institute. Some isolates displayed antibiotic resistance to the latest-generation fluoroquinolones (i.e., ciprofloxacin, levofloxacin, and norfloxacin) in vitro. Therefore, we screened for genes conferring resistance to fluoroquinolones and performed conjugation experiments to establish the resistance mechanisms. The antibiotic resistance rates were 14.29-21.42% to three kinds of fluoroquinolones: ciprofloxacin, levofloxacin, and norfloxacin. Among the 42 strains isolated, 15 carried the qnrS2 gene. The MICs of the fluoroquinolones in transconjugants with qnrS2 were more than fourfold higher compared with the recipient. Among the fluoroquinolone-resistant A. veronii strains, eight had point mutations in both gyrA codon 83 (Ser83→Ile83) and parC codon 87 (Ser87→Ile87). However, five isolates with point mutations in parC codon 52 remained susceptible to the three fluoroquinolones. In conclusion, the mechanisms of fluoroquinolone resistance in A. veronii isolates may be related to mutations in gyrA codon 83 and parC codon 87 and the presence of the qnrS2 gene.

OmpN, outer membrane proteins of Edwardsiella ictaluri are potential vaccine candidates for channel catfish (Ictalurus punctatus).

Outer membrane proteins (OMPs) are a class of proteins that reside in the outer membrane of Gram-negative bacteria. OMPs act as epitopes and are potential vaccine candidates. Outer membrane protein N (OmpN) is a component of the outer membrane of Edwardsiella ictaluri (E. ictaluri). In a previous study, the OmpN1-, OmpN2-, OmpN3-encoding genes of E. ictaluri were cloned, and here they were expressed in Escherichia coli. Western blotting showed that these three proteins had molecular weights of ∼60kDa. Channel catfish were immunized with recombinant OmpNs (rOmpNs) and then challenged with E. ictaluri. The results showed that rOmpN1, rOmpN2, and rOmpN3, as well as a mixture of all three proteins (in a ratio of 1:1:1) generated moderate immune protection (relative percentage of survival=62.5, 62.5, 67.5, and 75%, respectively). In an agglutination antibody titer assay, fish antisera showed an antibody titer of 1:128. Furthermore, each of the proteins stimulated high levels of lysozyme activity. In addition, a real-time polymerase chain reaction analysis revealed significant up-regulation of immune-related genes encoding major histocompatibility complex class I (MHC I), MHC II, CD4L, tumor necrosis factor-α, and interferon-γ after 24 and 48h of challenge, compared with the levels stimulated by phosphate-buffered saline. Taken together, we conclude that rOmpNs may elicit immune responses and generate protection against E. ictaluri in channel catfish. Thus, rOmpNs could be promising vaccine candidates against E. ictaluri.

A recombinant truncated surface immunogenic protein (tSip) plus adjuvant FIA confers active protection against Group B streptococcus infection in tilapia.

PURPOSE: Tilapia is an important agricultural fish that has been plagued by Group B streptococcus (GBS) infections in recent years, some of them severe. It is well-known that surface immunogenicity protein (Sip) is an effective vaccine against GBS. EXPERIMENTAL DESIGN: Since Sip was not expressed in either E. coli BL21 or E. coli Rosetta, we removed the N-terminal signal peptide and LysM of the virus to produce purified truncated Sip (tSip(1)), which multiplied easily in an E. coli host. The antibody's ability to recognize and combine with GBS was determined by Western-blot and specific staining in vitro. The relative percentage of survival (RPS), antibody titers, bacterial recovery, and pathologic morphology were monitored in vivo to evaluate the immune effects. Freund's incomplete adjuvant (FIA) plus tSip and aluminum hydroxide gel (AH) plus tSip were also evaluated. RESULTS: It revealed that tSip mixed with FIA was an effective vaccine against GBS in tilapia, while AH is toxic to tilapia.